Spatial Sequencing of Nucleic Acids Using DNA Origami Probes

ABSTRACT

A method of sequencing nucleic acids is provided using DNA origami as a barcode for a nucleic acid probe.

RELATED APPLICATION

This application is a continuation application which claims priority toU.S. patent application Ser. No. 15/886,163, filed Feb. 1, 2018, whichis a continuation application which claims priority to U.S. patentapplication Ser. No. 14/402,795, filed on Nov. 21, 2014, which is aNational Stage Application under 35 U.S.C. 371 of PCT applicationPCT/US2013/044241 designating the United States and filed Jun. 5, 2013;which claims the benefit of U.S. provisional application No. 61/655,528and filed Jun. 5, 2012 each of which are hereby incorporated byreference in their entireties.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with Government support under NIH Grant Number5P50HG005550-02. The Government has certain rights in the invention.

FIELD

The present invention relates to methods of sequencing nucleic acids.

BACKGROUND

Sequencing methods are known. See for example Shendure et al., Accuratemultiplex polony sequencing of an evolved bacterial genome, Science,vol. 309, p. 1728-32. 2005; Drmanac et al., Human genome sequencingusing unchained base reads on self-assembling DNA nanoarrays, Science,vol. 327, p. 78-81. 2009; McKernan et al., Sequence and structuralvariation in a human genome uncovered by short-read, massively parallelligation sequencing using two-base encoding, Genome Res., vol. 19, p.1527-41. 2009; Rodrigue et al., Unlocking short read sequencing formetagenomics, PLoS One, vol. 28, e11840. 2010; Rothberg et al., Anintegrated semiconductor device enabling non-optical genome sequencing,Nature, vol. 475, p. 348-352. 2011; Margulies et al., Genome sequencingin microfabricated high-density picolitre reactors, Nature, vol. 437, p.376-380. 2005; Rasko et al. Origins of the E. coli strain causing anoutbreak of hemolytic-uremic syndrome in Germany, N. Engl. J. Med.,Epub. 2011; Hutter et al., Labeled nucleoside triphosphates withreversibly terminating aminoalkoxyl groups, Nucleos. Nucleot. Nucl.,vol. 92, p. 879-895. 2010; Seo et al., Four-color DNA sequencing bysynthesis on a chip using photocleavable fluorescent nucleotides, Proc.Natl. Acad. Sci. USA., Vol. 102, P. 5926-5931 (2005); Olejnik et al.;Photocleavable biotin derivatives: a versatile approach for theisolation of biomolecules, Proc. Natl. Acad. Sci. U.S.A., vol. 92, p.7590-7594. 1995; U.S. Pat. No. 5,750,34; US 2009/0062129 and US2009/0191553.

SUMMARY

Embodiments of the present disclosure are directed to methods fordetermining the sequence of nucleotides in a target polynucleotide usingsequencing by ligation and/or sequencing by hybridization. Certainaspects include hybridizing and/or ligating a probe having a spatiallydistinct nucleic acid structure to a nucleic acid template, such as asingle stranded nucleic acid template. According to one aspect, thespatially distinct nucleic acid structure is representative of one ormore or all of the nucleotides in the oligonucleotide probe. Accordingto one aspect, the spatially distinct nucleic acid structure isrepresentative of a plurality of the nucleotides in the oligonucleotideprobe. According to one aspect, the spatially distinct nucleic acidstructure is representative of all of the nucleotides in theoligonucleotide probe.

Embodiments of the present disclosure are directed to methods fordetermining the sequence of nucleotides in a target polynucleotide usingsequencing by ligation and/or sequencing by hybridization. Certainaspects include hybridizing and/or ligating a probe having anon-naturally occurring two dimensional or three dimensional nucleicacid structure to a nucleic acid template, such as a single strandednucleic acid template. According to one aspect, the non-naturallyoccurring two dimensional or three dimensional nucleic acid structure isrepresentative of one or more or all of the nucleotides in theoligonucleotide probe. According to one aspect, the non-naturallyoccurring two dimensional or three dimensional nucleic acid structure isrepresentative of a plurality of the nucleotides in the oligonucleotideprobe. According to one aspect, the non-naturally occurring twodimensional or three dimensional nucleic acid structure isrepresentative of all of the nucleotides in the oligonucleotide probe.

Embodiments of the present disclosure are directed to methods fordetermining the sequence of nucleotides in a target polynucleotide usingsequencing by ligation and/or sequencing by hybridization. Certainaspects include hybridizing and/or ligating a probe having a visuallyresolvable or visually distinguishable two dimensional or threedimensional nucleic acid structure to a nucleic acid template, such as asingle stranded nucleic acid template. According to one aspect, thevisually resolvable or visually distinguishable two dimensional or threedimensional nucleic acid structure is representative of one or more orall of the nucleotides in the oligonucleotide probe. According to oneaspect, the visually resolvable or visually distinguishable twodimensional or three dimensional nucleic acid structure isrepresentative of a plurality of the nucleotides in the oligonucleotideprobe. According to one aspect, the visually resolvable or visuallydistinguishable two dimensional or three dimensional nucleic acidstructure is representative of all of the nucleotides in theoligonucleotide probe.

Embodiments of the present disclosure are directed to methods fordetermining the sequence of nucleotides in a target polynucleotide usingsequencing by ligation and/or sequencing by hybridization. Certainaspects include hybridizing and/or ligating a probe having a nucleicacid origami structure to a nucleic acid template, such as a singlestranded nucleic acid template. According to one aspect, the nucleicacid origami structure is representative of one or more or all of thenucleotides in the oligonucleotide probe. According to one aspect, thenucleic acid origami structure is representative of a plurality of thenucleotides in the oligonucleotide probe. According to one aspect, thenucleic acid origami structure is representative of all of thenucleotides in the oligonucleotide probe.

According to certain aspects, spatially distinct nucleic acidstructures, non-naturally occurring two dimensional or three dimensionalnucleic acid structures, and visually resolvable or visuallydistinguishable two dimensional or three dimensional nucleic acidstructures may be collectively referred to herein as nucleic acidorigami structures.

According to certain aspects, a set of probes can be designed withnucleic acid origami structures being geometrically distinct orgeometrically unique within the set of probes such that the nucleic acidorigami structure corresponds to a known one or more or all of thenucleotides in a particular oligonucleotide probe. In this manner, thegeometrically distinct or geometrically unique nucleic acid origamistructure is a barcode for the one or more or all of the nucleotides ina particular oligonucleotide probe. By determining the structure of thegeometrically distinct or geometrically unique nucleic acid origamistructure, the corresponding one or more or all of the nucleotides inthe oligonucleotide probe can be identified. According to one aspect,the nucleic acid origami structure is visually distinct. According toone aspect, the nucleic acid origami structure is spatially distinct.Since the one or more or all of the nucleotides in the oligonucleotideprobe can be identified, the corresponding complementary one or more orall of the nucleotides in the nucleic acid template can be identified.Using a plurality of oligonucleotide probes having geometricallydistinct or geometrically unique nucleic acid origami structures whichcan be identified, the sequence of the nucleotides in the nucleic acidtemplate can be determined using sequencing by ligation methods or usingsequencing by hybridization methods.

According to one aspect, the nucleic acid origami structure may includeone or more detectable moieties or barcodes. According to this aspect,the one or more detectable moieties or barcodes correspond to a knownone or more or all of the nucleotides in a particular oligonucleotideprobe. One such nucleotide is the terminal hybridized nucleotide in theoligonucleotide probe. According to this exemplary aspect, a set ofoligonucleotide probes include an A, C, G, or T as the terminalhybridized nucleotide with a different detectable moiety or barcodecorresponding to one of A, C, G, or T. Since the detectable moiety orbarcode corresponds to a known nucleotide, detection of the detectablemoiety or barcode confirms hybridization and/or ligation of a particularoligonucleotide probe from within a set of oligonucleotide probes andthe identity of the terminal hybridized nucleotide of theoligonucleotide probe. In addition, certain embodiments of the presentdisclosure discussed herein utilize different detectable moieties orbarcodes capable of identifying two or more different oligonucleotidesat the same time, as the different detectable moieties or barcodes arecapable of differentiating between different oligonucleotides. As anexample, detectable moieties may have wavelengths different enough to bedistinguishable. Different barcodes may be differentiated ordistinguished using methods known to those of skill in the art.According to this aspect, as many oligonucleotides could be identifiedas there are distinguishable detectable moieties or barcodes. Accordingto an additional aspect, a particular detectable moiety or barcode canrepresent one or more or all of the oligonucleotides in anoligonucleotide probe. Accordingly, the use of a detectable moiety orbarcode may be used for any nucleotide or combination of nucleotideswithin the oligonucleotide probe and is not limited to the terminalhybridized nucleotide.

Accordingly, one or more detectable moieties or barcodes can be attachedto a single nucleic acid origami structure or geometrically non-distinctor geometrically non-unique nucleic acid origami structure serving as ascaffold for the one or more detectable moieties or barcodes. Thedetectable moiety or barcode can be detected or identified. According toone aspect, the one or more detectable moieties or barcodes attached toa nucleic acid origami structure may render the nucleic acid origamistructure unique within a set of probes. For example, a uniquecombination of detectable moieties or detectable moieties with a uniqueplacement on the nucleic acid origami structure can allow the nucleicacid origami structure to uniquely identify a known one or more or allof the nucleotides in an oligonucleotide probe. In this manner, only asingle nucleic acid origami structure need be prepared for each of theoligonucleotide probes in a set since a unique barcode or uniquedetectable moiety or combination of detectable moieties or uniquelocation of a detectable moiety or moieties attached to each nucleicacid origami structure within the probe set will render the nucleic acidorigami structure uniquely representative for a known one or more or allof the nucleotides in a particular oligonucleotide probe. By identifyingthe one or more detectable moieties or barcodes attached to the nucleicacid origami structure, the corresponding one or more or all of thenucleotides in the oligonucleotide probe can be identified. Since theone or more or all of the nucleotides in the oligonucleotide probe canbe identified, the corresponding complementary one or more or all of thenucleotides in the nucleic acid template can be identified. Using aplurality of oligonucleotide probes having nucleic acid origamistructures with one or more detectable moieties or barcodes, thesequence of the nucleotides in the nucleic acid template can bedetermined using sequencing by ligation methods or using sequencing byhybridization methods.

According to certain aspects, one or more or all of the nucleotides in aparticular oligonucleotide probe can be identified using geometricallydistinct or geometrically unique nucleic acid origami structures ordetectable moieties or barcodes or a combination thereof. In thismanner, nucleic acid origami structures need not be geometricallydistinct or geometrically unique for a given set of probes. A singlenucleic acid origami structure design or a plurality of nucleic acidorigami structure designs can be used with unique barcodes or detectablemoieties to identify corresponding known one or more or all of thenucleotides in a particular oligonucleotide probe.

The nucleic acid origami structure may include a probe hybridizationsite corresponding to a known one or more or all of the nucleotides inthe oligonucleotide probe. A probe with a detectable moiety is thenhybridized to the probe hybridization site and the detectable moiety isdetected. According to certain aspects, a one or more or all of thenucleotides in the oligonucleotide probe is identified by detecting acorresponding detectable moiety and, accordingly, the correspondingcomplementary one or more or all of the nucleotides in the nucleic acidtemplate can be identified. According to one aspect, the nucleic acidorigami structure may include a probe hybridization site which can behybridized with a probe including a spatially detectable moiety. In thismanner, the probe with a spatially detectable moiety is a barcode forthe oligonucleotide probe.

According to still certain aspects, the nucleic acid origami structuremay be detached from the oligonucleotide probe and an additionaloligonucleotide probe having a nucleic acid origami structure may thenbe ligated and a one or more or all of the nucleotides in theoligonucleotide probe can be identified using the nucleic acid origamistructure as described herein. According to further aspects, anoligonucleotide probe may include a nucleic acid origami structureflanked on either side by an oligonucleotide probe suitable forhybridization and ligation. According to this aspect, a dual probestructure with a nucleic acid origami structure is provided.

According to a certain aspect, a nucleic acid template is provided forsequencing. The nucleic acid template may be either single stranded ordouble stranded. If single stranded, one or more sequencing primers maybe hybridized to the single stranded nucleic acid template. Thesequencing primers may be specific, semi-random or random. Thesequencing primers hybridized to the single stranded nucleic acidtemplate allow for hybridization and ligation of probes with nucleicacid origami structures. According to an additional embodiment, if thenucleic acid template is double stranded, one strand will serve as thetemplate and portions of the complementary strand will be removed usingmethods known to those of skill in the art and hybridized portions willremain which serve as sequencing primers. The hybridized portions of thecomplementary strand allow for hybridization and ligation ofoligonucleotide probes with nucleic acid origami structures.

According to a certain aspect, a nucleic acid template to be sequencedmay be stretched, straight or partially straight. For example, a nucleicacid template to be sequenced may be stretched into an elongatedposition, drawn out or elongated spatially, straightened or partiallystraightened. The nucleic acid template may be maintained in astretched, elongated, drawn out, straight or partially straight positionduring one or more steps of the sequencing methods described herein. Forexample, a single stranded nucleic acid template may be attached to asubstrate at either the 5′ or 3′ end and the nucleic acid template maybe stretched, stretched into an elongated position, drawn out, elongatedspatially, straightened or partially straightened under force, such as aphysical force to produce a nucleic acid template that is stretched,elongated, straight, or partially straight. One or more sequencingprimers are hybridized along the length of the stretched, elongated,straight or partially straight nucleic acid template. According to analternate aspect, one or more sequencing primers may be hybridized tothe nucleic acid template and then the nucleic acid template with thesequencing primers hybridized thereto may be stretched, elongated,straightened or partially straightened to produce a stretched,elongated, straight or partially straight nucleic acid templatesequencing primer construct. Methods of stretching or straighteningnucleic acids such as DNA are known. See for example Das et al., NucleicAcids Research, 2010, 1-8, doi:10.1093/nar/gkq673, Guan et al., PNAS,vol. 102, no. 51, pp. 18321-18325 (2005) and Allemand et al.,Biophysical Journal, vol. 73, pp. 2064-2070 (1997) (describing combingof DNA) each of which are hereby incorporated by reference in itsentirety.

One or more oligonucleotide probes having nucleic acid origamistructures are hybridized to the stretched, elongated, straight orpartially straight template nucleic acid sequence. According to analternate aspect, one or more oligonucleotide probes having nucleic acidorigami structures are hybridized to a template nucleic acid sequence,and then the construct is stretched, elongated, straightened orpartially straightened to form a stretched, elongated, straight orpartially straight template nucleic acid sequence and hybridized probeconstruct.

One or more oligonucleotide probes having nucleic acid origamistructures are hybridized to the straight or partially straight templatenucleic acid sequence and ligated to an adjacent sequencing primer.According to an alternate aspect, one or more oligonucleotide probeshaving nucleic acid origami structures are hybridized to a templatenucleic acid sequence and ligated to an adjacent sequencing primer toform a construct, and then the construct is stretched, elongated,straightened or partially straightened to form a stretched, elongated,straight or partially straight template nucleic acid sequence andhybridized and ligated probe construct.

According to one aspect, the template nucleic acid sequence is imaged orotherwise analyzed to identify the nucleic acid origami structures ordetectable moieties or barcodes attached thereto. In this manner, theknown one or more or all of the nucleotides in a particularoligonucleotide probe corresponding to a unique nucleic acid origamistructure or detectable moiety or barcode can be identified. Accordingto an additional aspect, the stretched, elongated, straight or partiallystraight template nucleic acid sequence with the oligonucleotide probeshaving nucleic acid origami structures is imaged or otherwise analyzedto identify the nucleic acid origami structures or detectable moietiesor barcodes attached thereto. In this manner, the known one or more orall of the nucleotides in a particular oligonucleotide probecorresponding to a unique nucleic acid origami structure or detectablemoiety or barcode can be identified.

According to the present disclosure, cycles of ligation and detectionmay be carried out along the length of the template nucleic acid ineither the 5′ to 3′ direction or the 3′ to 5′ direction. Then, theprocess may be repeated starting again from either the 5′ or 3′direction to identify additional nucleotides in the template nucleicacid. The process may be repeated until some, a plurality or all of thenucleotides in the template nucleic acid are identified as desired.According to an additional aspect, cycles of ligation and detection maybe carried out in both the 5′ to 3′ direction and the 3′ to 5′ directionin parallel. According to one aspect, nucleotides may be identified as aresult of ligations at the 5′ end or as a result of ligations at the 3′end or both.

According to one aspect, a sequencing primer is hybridized to a singlestranded nucleic acid template. An oligonucleotide probe is hybridizedto the single stranded nucleic acid template and ligated to thesequencing primer to form an extended hybridized sequence. According toone aspect of the present disclosure, the oligonucleotide probe includesa nucleic acid origami structure. One feature of the nucleic acidorigami structure is that it may prevent or inhibit or block multipleligations of the oligonucleotide probe such that a single ligationoccurs in a single cycle. Another feature of the nucleic acid origamistructure is that it facilitates perfectly matched hybridization of theoligonucleotide probe as the nucleic acid origami structure includes anucleotide directly attached to the oligonucleotide probe and where suchnucleotide does not hybridize to the template nucleic acid. Accordingly,an additional feature of the nucleic acid origami structure is that itmay be immediately adjacent and/or attached to the terminal hybridizednucleotide in the oligonucleotide probe such that the oligonucleotideprobe is hybridized to the single stranded nucleic acid template whilethe nucleic acid origami structure is not. Such a combination of anoligonucleotide probe and nucleic acid origami structure reduces biasinsofar as the number of nucleotides in the oligonucleotide probehybridized to the template nucleic acid is fixed and in some embodimentsthe terminal hybridized nucleotide of the oligonucleotide probe isextendable for further ligation.

It is to be understood that according to some aspects, the nucleic acidorigami structure need not be adjacent and/or attached to the terminalhybridized nucleotide in the oligonucleotide probe. The nucleic acidorigami structure may be attached to the oligonucleotide probe by alinker molecule. Exemplary embodiments include the nucleic acid origamistructure adjacent and/or attached to one of the nucleotides within theoligonucleotide probe such that detection of the detectable moietyconfirms hybridization and/or ligation of a particular oligonucleotideprobe from within the set and the identity of the hybridized nucleotideof the oligonucleotide probe to which the nucleic acid origami structureis attached, as a particular nucleic acid origami structure may beassociated with a known particular A, C, G, or T of the hybridizednucleotide to which the template-nonhybridizing nucleic acid structureis attached.

According to a further exemplary embodiment, the nucleic acid origamistructure need not be adjacent and/or attached to the nucleotide it willidentify. For example, the nucleic acid origami structure may beadjacent and/or attached to the terminal hybridized nucleotide, but thenucleic acid origami structure or detectable moiety or barcode attachedthereto is indicative of a known A, C, G or T at a known position withinthe hybridized and/or ligated oligonucleotide probe. According to thisaspect, the oligonucleotide probe is designed with a particular nucleicacid origami structure indicative of a particular nucleotide at aparticular position along the oligonucleotide probe. Also, theoligonucleotide probe is designed with a particular nucleic acid origamistructure indicative of a subset or all of the nucleotides in theoligonucleotide probe.

As an exemplary aspect, a set of oligonucleotide probes having Nnucleotides is prepared including a nucleic acid origami structureadjacent and/or attached to one of the N nucleotides and indicative ofone of the N nucleotides at a particular position within theoligonucleotide probe, indicative of a plurality of nucleotides atparticular positions within the oligonucleotide probe or indicative ofall of the nucleotides in the oligonucleotide probe. The nucleic acidorigami structure may be a unique nucleic acid origami structure or itmay include one or more detectable moieties or barcodes uniquelyidentifying the desired nucleotides in the oligonucleotide probe.According to this aspect, a desired nucleotide anywhere within theoligonucleotide probe or plurality of desired nucleotides within theprobe or the entire sequence of the oligonucleotide probe may beidentified for a given cycle of hybridization/and or ligation andidentification and/or detection of the nucleic acid origami structure.

The nucleic acid origami structure may include one or more probehybridization sites for hybridizing with a probe including a detectablemoiety with each probe hybridization site corresponding to a nucleotidein the oligonucleotide probe. For example, the oligonucleotide probe mayinclude a nucleic acid origami structure with a probe hybridization sitecorresponding to the known terminal hybridized nucleotide in theoligonucleotide probe. Hybridizing a probe with a detectable moiety tothe probe hybridization site on the nucleic acid origami structure anddetecting the detectable moiety identifies the terminal hybridizednucleotide, and the corresponding complementary nucleotide in thetemplate nucleic acid.

The nucleic acid origami structure may include a plurality of probehybridization sites with each probe hybridization site corresponding toa particular nucleotide at a particular position in the oligonucleotideprobe. According to this aspect, for an oligonucleotide of Nnucleotides, the nucleic acid origami structure may have N probehybridization sites, with each probe hybridization site corresponding toa specific nucleotide at a specific location along the oligonucleotideprobe. According to an additional aspect, for an oligonucleotide of Nnucleotides, the nucleic acid origami structure may have N or fewerprobe hybridization sites, with each probe hybridization sitecorresponding to a specific nucleotide at a specific location along theoligonucleotide probe. According to this aspect, the oligonucleotideprobe may include a nucleic acid origami structure having 1, 2, 3, 4, 5,or 6, etc., or up to N probe hybridization sites such that theoligonucleotide probe can be used to detect one nucleotide, Nnucleotides or fewer than N nucleotides. Embodiments of the presentdisclosure include the use of probes described herein for detecting andidentifying a plurality of nucleotides, such as two nucleotides, morethan two nucleotides, more than three nucleotides, more than 4nucleotides, more than 5 nucleotides, more than 6 nucleotides, etc. inan oligonucleotide probe as a result of a single ligation step or cycle.

According to one aspect of the present disclosure, a nucleic acidorigami structure is cleavably attached to the oligonucleotide probe.The nucleic acid origami structure has a cleavable nucleotideimmediately attached to a terminal hybridized nucleotide of theoligonucleotide probe. The cleavable nucleotide may be part of a doublestranded portion of the nucleic acid origami structure which isconnected to a geometrically distinct or geometrically unique structure.

According to this aspect, such a combination promotes cleavage at thedesired cleavage site leaving a precise oligonucleotide probe of knownlength thereby reducing bias. According to an additional aspect, anoptional step is provided of removing the nucleic acid origami structureby cleavage of the cleavable nucleotide and generating an extendableterminus on the extended hybridized sequence. According to one aspect,the step of cleaving can generate an extendable terminus available forligation. According to an alternate aspect, a nonextendable terminus ofthe oligonucleotide probe can be modified to be an extendable terminusavailable for ligation. According to this aspect, additionaloligonucleotide probes can then repeatedly be hybridized and ligated inseries along the single stranded nucleic acid template wherein aftereach ligation, one or more or all of the nucleotides of the hybridizedand ligated oligonucleotide probe are identified and one or more or allof the complementary nucleotides in the template nucleic acid areidentified.

In order to sequence each nucleotide in the nucleic acid template, theligation and/or hybridization methods described herein may be repeatedalong the length of the nucleic acid template and then the methodsrepeated one or more nucleotides out of phase along the length of thenucleic acid template compared to the sequencing method previouslyperformed. In this manner, where a single nucleotide is identified usingan oligonucleotide primer of N nucleotides (as an example), ligationand/or hybridization is repeated N−1 times one nucleotide out of phase.

Stated differently, the starting nucleotide in each successivesequencing method is out of phase by one nucleotide thereby allowing theidentification of successive nucleotides of the template nucleic acid.

According to an additional aspect of the methods of the presentdisclosure, a dual probe is provided that includes a firstoligonucleotide probe, a nucleic acid origami structure and a secondoligonucleotide probe. According to one aspect, the nucleic acid origamistructure is intermediate the first oligonucleotide probe and the secondoligonucleotide probe such that the first oligonucleotide probe and thesecond oligonucleotide probe may hybridize to the nucleic acid templatewith the nucleic acid origami structure therebetween.

A sequencing primer is hybridized to a nucleic acid template. The firstoligonucleotide probe and the second oligonucleotide probe of the dualprobe each hybridize to the single stranded nucleic acid template withthe first oligonucleotide probe being ligated to the sequencing primerto form an extended hybridized sequence. According to one aspect, thenucleic acid origami structure is used to identify one or more or all ofthe nucleotides in one or both of the first oligonucleotide probe or thesecond oligonucleotide probe.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1 is a schematic depicting hybridization and ligation of threeoligonucleotide probes to a DNA template with each oligonucleotide probehaving a geometrically distinct nucleic acid structure representative ofa known probe sequence.

FIG. 2 is a schematic depicting hybridization and ligation of twooligonucleotide probes to a DNA template with each oligonucleotide probehaving a geometrically similar nucleic acid structure but with eachhaving a set of detectable moieties acting as a barcode for a knownprobe sequence.

FIG. 3 is a schematic representation of 3D barrel shaped geometricallydistinct or spatially distinct nucleic acid structure, i.e. DNA origamistructure, barcoded with fluorescent particles.

FIGS. 4A-4C are a series of TEM images of geometrically distinct threedimensional nucleic acid origami structures and shapes, with and withoutdetectable gold nanoparticle probes (scale bar 20 nm).

FIG. 5 is an image of stretched DNA stained with YOYO-1, captured byfluorescent microscopy (blue channel) at 320× magnification.

FIG. 6 is a 3D CAD representation of a barrel-shaped Origami, with ahanging ssDNA binding site (left side), designed using cadnano.

FIG. 7 is an image of Origami probes bound to an aminosilane-coatedcover slip in the absence of template, captured by fluorescentmicroscopy at 320× magnification and blue, green and red channelssuperposed.

FIGS. 8A-8C are images of a set of stretched DNA templates, bound to avinylsilane-coated coverslip, assembled to a flow cell, and then probedwith fluorescently labeled Origami. The images were captured byfluorescent microscopy at 320× magnification. FIG. 8A: Green channel(CY3), FIG. 8B: Red channel (CY5), FIG. 8C: superposition of bothchannels.

FIG. 9 are images of stretched DNA template, bound to avinylsilane-coated coverslip, assembled to a flow cell, and then probedwith a set of fluorescently labeled Origami, each specific to adifferent nucleic acid base, using T4 DNA ligase. The image was capturedby fluorescent microscopy at 320× magnification. From left to right:Blue channel (FAM), Green channel (CY3), Red channel (CY5), andsuperposition of all three channels.

DETAILED DESCRIPTION

The principles of the present invention may be applied with particularadvantage to determine the identity of oligonucleotide sequences. Termsand symbols of nucleic acid chemistry, biochemistry, genetics, andmolecular biology used herein follow those of standard treatises andtexts in the field, e.g., Komberg and Baker, DNA Replication, SecondEdition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, SecondEdition (Worth Publishers, New York, 1975); Strachan and Read, HumanMolecular Genetics, Second Edition (Wiley-Liss, New York, 1999);Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach(Oxford University Press, New York, 1991); Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like.

Aspects of the present disclosure include the use of nucleic acidorigami structures in sequencing procedures. Nucleic acid origamistructures, also referred to as DNA origami structures or DNA origami,are two dimensional or three dimensional arbitrary shapes formed fromnucleic acids. The DNA origami may be non-naturally occurring nucleicacid nanostructures of arbitrary two dimensional or three dimensionalshape. In general, a non-naturally occurring nucleic acid nanostructureof arbitrary two dimensional or three dimensional shape can be made byfolding a single stranded nucleic acid scaffold into a custom shape andusing oligonucleotide strands to hybridize with the folded singlestranded nucleic acid scaffold and hold it into a custom shape. Thestructure of a DNA origami may be any arbitrary structure as desired.The DNA origami may be attached to an oligonucleotide probe and used asa unique identifier of one or more or all of the nucleotides in theprobe to which the DNA origami is attached. According to one aspect, aunique DNA origami structure is attached to each oligonucleotide probein a set of probes used in sequencing methods. The sequence of eacholigonucleotide probe in the set is known and accordingly, the uniqueDNA origami structure is associated with a known oligonucleotide probesequence.

According to one aspect, the DNA origami structure is spatiallydistinct. According to one aspect, the DNA origami structure isgeometrically distinct. According to one aspect, the DNA origamistructure can be directly visualized using methods known to those ofskill in the art. According to one aspect, DNA origami may take the formof any desired shape whether two dimensional or three dimensional. Aunique DNA origami structure may be associated with a knownoligonucleotide probe sequence. Accordingly, a set of oligonucleotideprobes of known sequence with each having a known unique DNA origamistructure associate with each known sequence is provided. Therefore,according to certain aspects of the disclosure, a unique DNA origamistructure may be used as a barcode for a particular oligonucleotideprobe sequence. During a sequencing method, a unique DNA origamistructure may be identified and accordingly, the probe sequence to whichit is attached can also be identified. Since DNA origami can serve as aunique identifier or barcode, the DNA origami attached to anoligonucleotide sequence can identify, as desired, any one or pluralityor all of the nucleotides of the probe to which it is attached.

FIG. 1 depicts aspects of certain embodiments of the methods and probesdescribed herein. According to FIG. 1, a nucleic acid template isprovided. The nucleic acid template may include a single strandednucleic acid template, such as a single stranded DNA template which maybe stretched as indicated by the stretched ssDNA in FIG. 1. As shown inFIG. 1, one or more sequencing primers is hybridized to a sequencingprimer hybridization site. The sequencing primers are spaced apart adistance along the single stranded DNA template. Sequencing primers maybe between about 10 and about 20 nucleotides in length. The sequencingprimers may be random primers as shown in FIG. 1 by the sequence (SEQ IDNO:1) NNNNNNNNNNNNNNN. However, the sequencing primers may besemi-random primers.

The sequencing primers may be non-random to the extent that sequencingprimers are designed to hybridize with certain desired locations on thesingle stranded DNA template. According to one aspect, a set ofsequencing primers is contacted to the single stranded DNA template andsequencing primers hybridize at various locations along the length ofthe single stranded DNA template. The sequencing primers can be designedsuch that there is a desired length of DNA between each hybridizedsequencing primer.

With reference to any particular sequencing primer hybridization site,adjacent to the sequencing primer hybridization site on the nucleic acidtemplate is a first template nucleotide NTI followed by templatenucleotides NT2 to NT6. As shown in FIG. 1, oligonucleotide probereferred to as an “origami probe” is hybridized to the nucleic acidtemplate and includes 6 nucleotides which hybridize respectively to the6 corresponding template nucleotides NT1 to NT6. The nucleotide of theorigami probe adjacent to the terminal nucleotide of the sequencingprimer is ligated to the terminal nucleotide of the sequencing primer.Methods and materials for sequencing by ligation are known to those ofskill in the art and include those described in Shendure et al.,Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome,Science, vol. 309, p. 1728-32 (2005) hereby incorporated by reference inits entirety. Connected to the origami probe is a nucleic acid origamistructure (“DNAOM”) which is spatially distinct or geometricallydistinct, an example of which is shown as a two dimensional circularstructure. According to one aspect, the spatially distinct orgeometrically distinct nucleic acid structure is representative of aknown probe sequence. Other nucleic acid origami structures are shown asa two dimensional square and a two dimensional triangle. The nucleicacid origami structures include a double stranded nucleic acid stem. Thedouble stranded stem is connected to nucleotide N6 of theoligonucleotide or “origami” probe. According to one aspect, the doublestranded stem is connected to nucleotide N6 of the oligonucleotide probeby a cleavable nucleotide. The cleavable nucleotide may also be referredto as a cut site or cleaving site as the cleavable nucleotide is removedfrom the oligonucleotide probe. According to certain aspects, thenucleic acid origami structure may be attached to the probe by a spacermolecule or tether molecule or an extender molecule which is understoodherein to be a molecule capable of being attached to both the probe andthe nucleic acid origami structure and which distances the nucleic acidorigami structure from the probe. Such molecules can be double strandednucleic acids, carbon chains and other molecules known to those of skillin the art useful as spacer molecules or tether molecules. Exemplarymolecules useful as spacers, tethers, or extenders and the like includesingle stranded homopolymer nucleic acid sequences (such as betweenabout 20 and about 45 nucleotides, between about 25 and about 40nucleotides or such as about 36 nucleotides), alkyl chain extenders forexample of between about 10 and about 30 carbon atoms (such as betweenabout 15 and about 25 carbon atoms, such as about 18 carbon atoms),polyethylene glycol chains, tetrahydrofuran derivatives and the like.Exemplary single stranded homopolymer nucleic acid sequences include achain or sequence of 36 dTTP nucleotides.

As shown in FIG. 1, each geometrically distinct nucleic acid origamistructure is representative of the known nucleotides in the probe towhich it is attached. For example, the circular origami structure isrepresentative of the nucleic acid sequence GAACG in the probe GAACGN.The square origami structure is representative of the nucleic acidsequence CCAGT in the probe CCAGTN. The triangle origami structure isrepresentative of the nucleic acid sequence TTAGT in the probe TTAGTN.It is to be understood that FIG. 1 is merely an illustration of aspectsof the present disclosure and is not meant to be limiting in any way.According to certain aspects, a unique DNA origami structure can becreated for any known nucleic acid sequence in a probe.

For a set of probes with all combinations of 5 known nucleotides insequence, 4⁵ or 1024 probes can be created with each probe having aunique DNA origami structure corresponding to the known 5 base sequence.Accordingly, 1024 unique DNA origami structures are created. Each uniqueDNA origami structure is assigned to a specific known nucleotide probesequence and is attached to the specific known nucleotide probesequence. This technique can be used with any probe length and any knownnucleic acid sequence length within a probe.

The structure of the unique DNA origami is visually recognizable andtherefore distinguishable from other unique DNA origami shapes. Methodsof making unique DNA origami shapes of arbitrary design or desireddesign are described in Rothemund, “Folding DNA to Create NanoscaleShapes and Patterns”, Nature March 2006, p. 297-302, vol. 440;Rothemund, “Design of DNA Origami”, Proceedings of the InternationalConference of Computer-Aided Design (ICCAD) 2005; and U.S. Pat. No.7,842,793 each of which are hereby incorporated by reference in itsentirety.

According to an additional embodiment, a DNA origami structure mayinclude one or more detectable moieties at one or more locations withinor on the DNA origami structure. One or more detectable moieties can actas a barcode for a particular probe sequence. One or more detectablemoieties at one or more locations within or on the DNA origami can actas a barcode for a particular probe sequence. According to one aspect,the visually detectable spatial orientation of the DNA origami or theone or more detectable moieties at one or more locations within or onthe DNA origami, or both, can act as a barcode for a particular probesequence. According to an exemplary embodiment, DNA origami may encodedwith features specific to a particular nucleic acid probe sequence.Additionally, DNA origami may be tagged with metal nano-particles orfluorophores to enhance distinguishability when analyzed or imaged.Additionally, DNA origami may be tagged with metal nano-particles orfluorophores at distinct locations to enhance distinguishability whenanalyzed or imaged.

As shown in FIG. 1, the single stranded nucleic acid template isstretched, straight or partially straight. Methods of stretching orstraightening nucleic acids are known to those of skill in the art andinclude Chung et al., Biomimetic Self-templating SupramolecularStructures, Nature 478, p. 364-368 (2011); Bensimon et al., StretchingDNA with a Receding Meniscus: Experiments and Models, Physical ReviewLetters, v74 n23, p. 4754-4757 (1995); and Zimmermann et al., DNAstretching on Functionalized Gold Surfaces, Nucleic Acids Research, v22n3, p. 492-497 (1994) each of which are hereby incorporated by referencein their entireties.

According to one aspect, the single stranded DNA template can bestraightened or partially straightened. According to one aspect, thesingle stranded DNA template can be straightened or partiallystraightened prior to hybridization with sequencing primers and/orprobes. According to one aspect, the single stranded DNA template can bestraightened or partially straightened after hybridization withsequencing primers and/or probes.

According to one aspect, the single stranded DNA template can bestraightened or partially straightened using shear flow with the singlestranded DNA template in buffer solution. Shear flow can be created byflowing fluid through a channel as with a flowcell or through the dryingof a meniscus. In addition, DNA can be straightened or partiallystraightened by attaching the DNA to a needle and pulling the DNA asdescribed in WO2009/046445, paragraph 147 and FIGS. 7, 8 and 15 herebyincorporated by reference herein. According to one aspect, the singlestranded DNA template is straightened on a substrate that can aid inimaging, such as a copper EM grid, a glass slide, or silicon wafer. Thesubstrates should be treated to allow the single stranded DNA templateto be attached to the surface of the substrate, such as by aminosilanetreatment in the case of a glass or silicon substrate or glow dischargeas is the case with an EM grid. Also, prior to straightening, a singleend of the single stranded DNA template (either 5′ or 3′ end) may befunctionalized and attached to the substrate to allow more accuratepositional control of the straightened DNA. Additional methods ofattaching DNA to substrates are known to those of skill in the art.

According to one aspect, straightening or partially straightening thenucleic acid template facilitates detection of the DNA origami as thetwo dimensional DNA origami or the three dimensional DNA origamistructure can be more easily distinguished from the straight orpartially straight nucleic acid template. According to a certain aspect,the DNA origami can include larger and more visually recognizabledistinct features thereby providing a size gain or a magnetic gainthrough field distortion.

According to one aspect, a scanning instrument can be used to visualizeand distinguish nucleic acid origami structures. In an exemplaryembodiment, the scanning instrument is an electron microscope. Exemplaryelectron microscopes include a transmission electron microscope (TEM), ascanning electron microscope (SEM), a scanning transmission electronmicroscope (STEM), and environmental scanning electron microscope(ESEM), a cryo-electron microscope (cryo-EM) and other electronmicroscopes known to those of skill in the art which can be used toidentify local DNA conformation. Transmission electron microscopes andmethods of using TEMs are known to those of skill in the art. See Morel,“Visualization of Nucleic Acids,” The Spreading of Nucleic Acids, p.35-56, CRC Press, Boca Raton (1995) hereby incorporated by reference inits entirety. According to one aspect, the nucleic acid template and thehybridized oligonucleotide probes with the DNA origami motifs arevisible to the nanometer scale. The EM scanning system scans along thelength of the linear or straight or partially straight construct of thenucleic acid template and the hybridized oligonucleotide probes with theDNA origami motifs. According to one aspect, image processing, edgedetection, and object recognition algorithms (such as the Sobelalgorithm) can be used to detect the end points and direction vector ofthe nucleic acid template, and inform the motion of the stage. Theconstruct of the nucleic acid template and the hybridizedoligonucleotide probes with the DNA origami motifs are stained withheavy metal for EM imaging.

According to additional aspects, the construct of the nucleic acidtemplate and the hybridized oligonucleotide probes with the DNA origamimotifs may be analyzed by methods known to those of skill in the artincluding high spatial resolution microscopy or super resolutionmicroscopy such as stochastic optical reconstruction microscopy (STORM).Other stochastic methods include spectral precision distance microscopy(SPDM), photoactivated localization microscopy (PALM). Additionalmethods include deterministic methods such as stimulated emissiondepletion (STED), ground state depletion (GSD) and spatially structuredillumination microscopy (SSIM). Still additional methods includescanning probe microscopy such as atomic force microscopy or scanningtunneling microscopy (STM), as well as, magnetic particles and amagnetic pickup, similar to a hard disk drive head.

FIG. 2 depicts an aspect of FIG. 1 where the nucleic acid origamistructures attached to the probes are similar or of the same structure.According to the embodiment of FIG. 2, each DNA origami structureincludes one or more detectable moieties which uniquely distinguish eachDNA origami structure. As shown in FIG. 2, the DNA origami structuresdiffer in a detectable label so that the two DNA origami structures canbe distinguished. According to one aspect, a DNA origami structure canact as a scaffold for the attachment of one or more detectable moietiesor barcodes such that the DNA origami construct can serve as a barcodefor the probe to which it is attached. According to this aspect, uniqueDNA origami constructs can be created for each unique probe in a probeset. For example, for a 5 base probe sequence, 4⁵ or 1024 unique DNAorigami constructs can be prepared to uniquely barcode each probesequence within the set of 1024 probes.

FIG. 3 depicts a fluorescent barcoded DNA origami structure. As can beseen, different detectable moieties (fluorophores) can be attached todifferent locations on the DNA origami structure to create a unique DNAorigami construct.

FIGS. 4A-4C depict different DNA origami shapes useful in the presentdisclosure. Some of the DNA origami structures are tagged with goldprobes at various locations on the DNA origami shapes. Some of the DNAorigami structures are tagged with antibody fragments at variouslocations on the DNA origami shapes.

Target Polynucleotides

Target polynucleotides, also referred to as oligonucleotides or templateoligonucleotides, to be sequenced according to the methods describedherein can be prepared in a variety of ways known to those of skill inthe art. According to one aspect, target polynucleotides are singlestranded nucleic acids. The length of the target polynucleotide canvary. According to certain aspects, the length of the targetpolynucleotide can be between about 1 nucleotide to about 3,000,000nucleotides in length, between about 1 nucleotide to about 2,500,000nucleotides in length, between about 1 nucleotide to about 2,000,000nucleotides in length, between about 1 nucleotide to about 1,500,000nucleotides in length, between about 1 nucleotide to about 1,000,000nucleotides in length, between about 1 nucleotide to about 500,000nucleotides in length, between about 1 nucleotide to about 250,000nucleotides in length, between about 1 nucleotide to about 200,000nucleotides in length or between about 1 nucleotide to about 150,000nucleotides in length. Exemplary target polynucleotide can be betweenabout 1 nucleotide to about 100,000 nucleotides in length, between about1 nucleotide to about 10,000 nucleotides in length, between about 1nucleotide to about 5,000 nucleotides in length, between about 4nucleotides to about 2,000 nucleotides in length, between about 6nucleotides to about 2,000 nucleotides in length, between about 10nucleotides to about 1,000 nucleotides in length, between about 20nucleotides to about 100 nucleotides in length, and any range or valuein between whether overlapping or not.

A template for sequencing can be prepared from several linear orcircular sources of polynucleotides, such as dsDNA, ssDNA, cDNA, RNA andsynthesized or naturally occurring oligonucleotides.

An exemplary template is a synthesized oligonucleotide of the form (SEQID NO:2) 5′-PO4-GTT CCT CAT TCT CTG AAG ANN NNN NNN NNN NNN NNN NNN NNNNNN NNN NNN NNN NAC TTC AGC TGC CCC GG-3′-OH, where the N portionrepresents a ssDNA template to be identified, (SEQ ID NO:3) GTT CCT CATTCT CTG AAG A and (SEQ ID NO:4) AC TTC AGC TGC CCC GG represent adaptersthat will be used as a sequencing primer hybridization site (PS1).According to aspects of the present disclosure, sequencing can beaccomplished in either the 5′ to 3′ direction or the 3′ to 5′ directionor both directions simultaneously. According to certain aspects,multiple copies of the template nucleic acid are prepared using methodsknown to those of skill in the art. According to one aspect, the ssDNAtemplate can be circularized using ssDNA Circligase II (Epicentre #CL9025K) or other ssDNA ligase such as Circligase I (Epicentre #CL4115K), or by template-directed ligation using a combination of adsDNA ligase (e.g. (T3, T4, T7 and other ds DNA ligases) with a bridgeoligo (SEQ ID NO:5) (5′-ATGAGGAACCCGGGGCAG-3′-PO₄). Chemical ligationmethods have also been described (Dolinnaya et al., 1993; Kumar et al.,2007).

According to one aspect, 10 pmol of ssDNA template is circularized usingCircligase II, according to the manufacturer's recommendation. Followingthe circularization, 20 units of Exonuclease I (Enzymatics # X801L) and100 units of Exonuclease III (Enzymatics # X802L) are added to thereaction to digest any remaining linear template. Next, rolling circleamplification (RCA) is performed on the circular ssDNA template using aDNA polymerase with high processivity, strong displacement activity andlow error rate. Rolling circle amplification methods are known to thoseof skill in the art and include Drmanac et al., Human genome sequencingusing unchained base reads on self-assembling DNA nanoarrays, Science,vol. 327, p. 78-81 (2009). According to one aspect, 1 pmol of thecircularized template is used with 20 units of phi29 DNA polymerase(Enzymatics # P702L). Additionally, dNTP (typically 1 mM) and a RCAprimer (typically 1 pmol) are required. An exemplary RCA primer wouldhave the form (SEQ ID NO:6) 5′-AATGAGGAACCCGGGGCA*G*C, where the *represents a phosphorothioate bond thereby indicating that the last 3′nucleotide bears a phosphorotioate bond, making the RCA less susceptibleto phi29 3′->5′ exonuclease activity. However, an exemplary RCA primermay not include such phosphorothioate bonds, especially if thepolymerase used does not have 3′->5′ exonuclease activity.Alternatively, an exemplary RCA primer may have phosphorothioate bondson the 5′ side of the RCA primer such as (SEQ ID NO:7)5′-A*A*TGAGGAACCCGGGGCAGC. An annealing reaction is often performedbefore adding the phi29 (95° C. for 1 min, then 2 min cool down to 4°C.), to increase the RCA efficiency. Then the reaction is incubated at30° C. for an hour (incubation periods between 15 min to 6 hours mayalso be used). Other temperatures can be used, since phi29 is activebetween 4° C. and 40° C. (with 90% diminished activity). Then, thereaction is cooled to 4° C. and the RCA products (referred to as Rolony)are recovered in cold PBS and can be stored at 4° C. until needed.Rolling circle amplification products prepared this way are stable forseveral months and can be used as template for assaying sequencingtechniques.

A template can also be prepared using dsDNA from a biological source.The genomic DNA would first be extracted using one of the severalextraction kits commercially available for that purpose. Then, the dsDNAwould be fragmented to any length or a specific length, using amechanical (Covaris focused electroacoustic, Nebulizer, sonication,vortex,) or enzymatic (e.g. Fragmentase) fragmentation. While, it may bepractical to keep the fragments size between 100 and 1000 nucleotides,any size can be used. For example, the length of the targetpolynucleotide template can be between about 1 nucleotide to about3,000,000 nucleotides in length, between about 1 nucleotide to about2,500,000 nucleotides in length, between about 1 nucleotide to about2,000,000 nucleotides in length, between about 1 nucleotide to about1,500,000 nucleotides in length, between about 1 nucleotide to about1,000,000 nucleotides in length, between about 1 nucleotide to about500,000 nucleotides in length, between about 1 nucleotide to about250,000 nucleotides in length, between about 1 nucleotide to about200,000 nucleotides in length or between about 1 nucleotide to about150,000 nucleotides in length. Exemplary target polynucleotide templatescan be between about 1 nucleotide to about 100,000 nucleotides inlength, between about 1 nucleotide to about 10,000 nucleotides inlength, between about 1 nucleotide to about 5,000 nucleotides in length,between about 4 nucleotides to about 2,000 nucleotides in length,between about 6 nucleotides to about 2,000 nucleotides in length,between about 10 nucleotides to about 1,000 nucleotides in length,between about 20 nucleotides to about 100 nucleotides in length, and anyrange or value in between whether overlapping or not. In certaininstances, the target polynucleotide may be an entire strand of genomicDNA or a portion or fragment thereof.

The ends of the fragmented dsDNA are repaired and phosphorylated in onestep using a mix of T4 DNA polymerase and T4 Polynucleotide Kinase(Enzymatics # Y914-HC-L), according to the manufacturer instructions.Other DNA polymerase with 3′->5′ exonuclease activity and low or nostrand displacement activity can be used. Adapters composed of dsDNAoligonucleotides are added to the dsDNA using a DNA ligase, typically T3(Enzymatics # L601L) or T4 DNA ligase (Enzymatics # L603-HC-L). Thereaction is performed at room temperature for 20 min according to themanufacturer instructions. The adapters can be in the form Ad1 (SEQ IDNO:8) 5′-GTTCCTCATTCTCTGAAGA, Ad2 (SEQ ID NO:9) 5′-TCTTCAGAGAATGAG, Ad3(SEQ ID NO:10) 5′-CCGGGGCAGCTGAAGT, and Ad4 (SEQ ID NO:11)5′-ACTTCAGCTGCC, where Ad1-Ad2 are annealed together and Ad3-Ad4 annealtogether, before being ligated. After ligation, the 5′ overhang ends arefilled-in using a DNA polymerase with, such as Bst DNA polymerase largefragment (NEB # M0275L). Next, limited PCR (typically 6 to 8 cycles) isperformed to generate multiple copies using PCR primer in the form (SEQID NO:12) 5′-PO4-GTTCCTCATTCTCTGAAGA and (SEQ ID NO:13)5′-Biotin-CCGGGGCAGCTGAAGT. The 5′biotin is then attached to one end ofthe dsDNA to streptavidin coated magnetic beads (Invitrogen #65305),allowing the other end to be recovered by performing the Circligase IIreaction, as described above, with the exception that the template isattached to the beads. This is performed by incubating the reaction at65° C. for 2 h, which will allow the DNA strand with 5′-PO4 to bede-anneal and be circularized. After exonuclease digest, the circularssDNA template is now ready for rolling circle amplification (RCA) asdiscussed above. Adapters can also be in the form Ad5 (SEQ ID NO:14)5′-GAAGTCTTCTTACTCCTTGGGCCCCGTCAGACTTC and Ad6 (SEQ ID NO:15)5′-GTTCCGAGATTTCCTCCGTTGTTGTTAATCGGAAC, where Ad5 and Ad6 each formhairpin structures to be ligated on each side of the dsDNA, virtuallycreating a circular ssDNA product ready for RCA. A pull down assay canbe used to select templates bearing one of each hairpin and not two ofthe same. In this case, an oligonucleotide complementary to one loop inthe form (SEQ ID NO:16) 5′-Biotin-TAACAACAACGGAGGAAA-C3 sp will be boundto streptavidin coated magnetic beads. Next RCA can be performed using aRCA primer (SEQ ID NO:17) (5′-ACGGGGCCCAAGGAGTA*A*G), as describedabove.

Other amplification methods can be used. In general, “amplifying”includes the production of copies of a nucleic acid molecule of thearray or a nucleic acid molecule bound to a bead via repeated rounds ofprimed enzymatic synthesis. “In situ” amplification indicated that theamplification takes place with the template nucleic acid moleculepositioned on a support or a bead, rather than in solution. In situamplification methods are described in U.S. Pat. No. 6,432,360.

Varied choices of polymerases exist with different properties, such astemperature, strand displacement, and proof-reading. Amplification canbe isothermal, as described above and in similar adaptation such asmultiple displacement amplification (MDA) described by Dean et al.,Comprehensive human genome amplification using multiple displacementamplification, Proc. Natl. Acad. Sci. U.S.A., vol. 99, p. 5261-5266.2002; also Dean et al., Rapid amplification of plasmid and phage DNAusing phi29 DNA polymerase and multiply-primed rolling circleamplification, Genome Res., vol. 11, p. 1095-1099. 2001; alsoAviel-Ronen et al., Large fragment Bst DNA polymerase for whole genomeamplification of DNA formalin-fixed paraffin-embedded tissues, BMCGenomics, vol. 7, p. 312. 2006. Amplification can also cycle throughdifferent temperature regiments, such as the traditional polymerasechain reaction (PCR) popularized by Mullis et al., Specific enzymaticamplification of DNA in vitro: The polymerase chain reaction. ColdSpring Harbor Symp. Quant. Biol., vole 51, p. 263-273. 1986. Variationsmore applicable to genome amplification are described by Zhang et al.,Whole genome amplification from a single cell: implications for geneticanalysis, Proc. Natl. Acad. Sci. U.S.A., vol. 89, p. 5847-5851. 1992;and Telenius et al., Degenerate oligonucleotide-primed PCR: generalamplification of target DNA by a single degenerate primer, Genomics,vol. 13, p. 718-725. 1992. Other methods include Polony PCR described byMitra and Church, In situ localized amplification and contactreplication of many individual DNA molecules, Nuc. Acid. Res., vole 27,pages e34. 1999; emulsion PCR (ePCR) described by Shendure et al.,Accurate multiplex polony sequencing of an evolved bacterial genome,Science, vol. 309, p. 1728-32. 2005; and Williams et al., Amplificationof complex gene libraries by emulsion PCR, Nat. Methods, vol. 3, p.545-550. 2006. Any amplification method can be combined with a reversetranscription step, a priori, to allow amplification of RNA. Accordingto certain aspects, amplification is not absolutely required sinceprobes, reporters and detection systems with sufficient sensitivity canbe used to allow detection of a single molecule using templatenon-hybridizing nucleic acid structures described. Ways to adaptsensitivity in a system include choices of excitation sources (e.g.illumination) and detection (e.g. photodetector, photomultipliers). Waysto adapt signal level include probes allowing stacking of reporters, andhigh intensity reporters (e.g. quantum dots) can also be used.

Amplification methods useful in the present disclosure may comprisecontacting a nucleic acid with one or more primers that specificallyhybridize to the nucleic acid under conditions that facilitatehybridization and chain extension. Exemplary methods for amplifyingnucleic acids include the polymerase chain reaction (PCR) (see, e.g.,Mullis et al. (1986) Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1:263and Cleary et al. (2004) Nature Methods 1:241; and U.S. Pat. Nos.4,683,195 and 4,683,202), anchor PCR, RACE PCR, ligation chain reaction(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; andNakazawa et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:360-364), selfsustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad.Sci. U.S.A. 87:1874), transcriptional amplification system (Kwoh et al.(1989) Proc. Natl. Acad. Sci. U.S.A. 86:1173), Q-Beta Replicase (Lizardiet al. (1988) BioTechnology 6:1197), recursive PCR (Jaffe et al. (2000)J. Biol. Chem. 275:2619; and Williams et al. (2002) J. Biol. Chem.277:7790), the amplification methods described in U.S. Pat. Nos.6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and 5,612,199, orany other nucleic acid amplification method using techniques well knownto those of skill in the art. In exemplary embodiments, the methodsdisclosed herein utilize PCR amplification.

In certain exemplary embodiments, methods for amplifying nucleic acidsequences are provided. Exemplary methods for amplifying nucleic acidsinclude the polymerase chain reaction (PCR) (see, e.g., Mullis et al.(1986) Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1:263 and Cleary etal. (2004) Nature Methods 1:241; and U.S. Pat. Nos. 4,683,195 and4,683,202), anchor PCR, RACE PCR, ligation chain reaction (LCR) (see,e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) Proc. Natl. Acad. Sci. U.S.A. 91:360-364), self sustainedsequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.U.S.A. 87:1874), transcriptional amplification system (Kwoh et al.(1989) Proc. Natl. Acad. Sci. U.S.A. 86:1173), Q-Beta Replicase (Lizardiet al. (1988) BioTechnology 6:1197), recursive PCR (Jaffe et al. (2000)J. Biol. Chem. 275:2619; and Williams et al. (2002) J. Biol. Chem.277:7790), the amplification methods described in U.S. Pat. Nos.6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and 5,612,199,isothermal amplification (e.g., rolling circle amplification (RCA),hyperbranched rolling circle amplification (HRCA), strand displacementamplification (SDA), helicase-dependent amplification (HDA), PWGA) orany other nucleic acid amplification method using techniques well knownto those of skill in the art.

“Polymerase chain reaction,” or “PCR,” refers to a reaction for the invitro amplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g., exemplified by the references:McPherson et al., editors, PCR: A Practical Approach and PCR 2: APractical Approach (IRL Press, Oxford, 1991 and 1995, respectively). Forexample, in a conventional PCR using Taq DNA polymerase, a doublestranded target nucleic acid may be denatured at a temperature greaterthan 90° C., primers annealed at a temperature in the range 50-75° C.,and primers extended at a temperature in the range 68-78° C.

The term “PCR” encompasses derivative forms of the reaction, includingbut not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR,multiplexed PCR, assembly PCR and the like. Reaction volumes range froma few hundred nanoliters, e.g., 200 nL, to a few hundred microliters,e.g., 200 μL. “Reverse transcription PCR,” or “RT-PCR,” means a PCR thatis preceded by a reverse transcription reaction that converts a targetRNA to a complementary single stranded DNA, which is then amplified,e.g., Tecott et al., U.S. Pat. No. 5,168,038. “Real-time PCR” means aPCR for which the amount of reaction product, i.e., amplicon, ismonitored as the reaction proceeds. There are many forms of real-timePCR that differ mainly in the detection chemistries used for monitoringthe reaction product, e.g., Gelfand et al., U.S. Pat. No. 5,210,015(“Taqman”); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627(intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecularbeacons). Detection chemistries for real-time PCR are reviewed in Mackayet al., Nucleic Acids Research, 30:1292-1305 (2002). “Nested PCR” meansa two-stage PCR wherein the amplicon of a first PCR becomes the samplefor a second PCR using a new set of primers, at least one of which bindsto an interior location of the first amplicon. As used herein, “initialprimers” in reference to a nested amplification reaction mean theprimers used to generate a first amplicon, and “secondary primers” meanthe one or more primers used to generate a second, or nested, amplicon.“Multiplexed PCR” means a PCR wherein multiple target sequences (or asingle target sequence and one or more reference sequences) aresimultaneously carried out in the same reaction mixture, e.g. Bernard etal. (1999) Anal. Biochem., 273:221-228 (two-color real-time PCR).Usually, distinct sets of primers are employed for each sequence beingamplified. “Quantitative PCR” means a PCR designed to measure theabundance of one or more specific target sequences in a sample orspecimen. Techniques for quantitative PCR are well-known to those ofordinary skill in the art, as exemplified in the following references:Freeman et al., Biotechniques, 26:112-126 (1999); Becker-Andre et al.,Nucleic Acids Research, 17:9437-9447 (1989); Zimmerman et al.,Biotechniques, 21:268-279 (1996); Diviacco et al., Gene, 122:3013-3020(1992); Becker-Andre et al., Nucleic Acids Research, 17:9437-9446(1989); and the like.

In general, target polynucleotides, template nucleotides, templatenon-hybridizing nucleic acids or probes described herein include theterms “nucleic acid molecule,” “nucleic acid sequence,” “nucleic acidfragment,” “oligonucleotide” and “polynucleotide” and are usedinterchangeably and are intended to include, but not limited to, apolymeric form of nucleotides that may have various lengths, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof. Differentpolynucleotides may have different three-dimensional structures, and mayperform various functions, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, an exon, an intron,intergenic DNA (including, without limitation, heterochromatic DNA),messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, smallinterfering RNA (siRNA), cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of a sequence, isolatedRNA of a sequence, nucleic acid probes, and primers. Oligonucleotidesuseful in the methods described herein may comprise natural nucleic acidsequences and variants thereof, artificial nucleic acid sequences, or acombination of such sequences.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

Examples of modified nucleotides include, but are not limited todiaminopurine, S²T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,2,6-diaminopurine and the like. Nucleic acid molecules may also bemodified at the base moiety (e.g., at one or more atoms that typicallyare available to form a hydrogen bond with a complementary nucleotideand/or at one or more atoms that are not typically capable of forming ahydrogen bond with a complementary nucleotide), sugar moiety orphosphate backbone. Nucleic acid molecules may also containamine-modified groups, such as aminoallyl-dUTP (aa-dUTP) andaminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent attachment ofamine reactive moieties, such as N-hydroxy succinimide esters (NHS).

Oligonucleotide sequences may be isolated from natural sources orpurchased from commercial sources. In certain exemplary embodiments,oligonucleotide sequences may be prepared using one or more of thephosphoramidite linkers and/or sequencing by ligation methods known tothose of skill in the art. Oligonucleotide sequences may also beprepared by any suitable method, e.g., standard phosphoramidite methodssuch as those described herein below as well as those described byBeaucage and Carruthers ((1981) Tetrahedron Lett. 22: 1859) or thetriester method according to Matteucci et al. (1981) J. Am. Chem. Soc.103:3185), or by other chemical methods using either a commercialautomated oligonucleotide synthesizer or high-throughput, high-densityarray methods known in the art (see U.S. Pat. Nos. 5,602,244, 5,574,146,5,554,744, 5,428,148, 5,264,566, 5,141,813, 5,959,463, 4,861,571 and4,659,774, incorporated herein by reference in its entirety for allpurposes). Pre-synthesized oligonucleotides may also be obtainedcommercially from a variety of vendors.

In certain exemplary embodiments, oligonucleotide sequences may beprepared using a variety of microarray technologies known in the art.Pre-synthesized oligonucleotide and/or polynucleotide sequences may beattached to a support or synthesized in situ using light-directedmethods, flow channel and spotting methods, inkjet methods, pin-basedmethods and bead-based methods set forth in the following references:McGall et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:13555; SyntheticDNA Arrays In Genetic Engineering, Vol. 20:111, Plenum Press (1998);Duggan et al. (1999) Nat. Genet. S21:10; Microarrays: Making Them andUsing Them In Microarray Bioinformatics, Cambridge University Press,2003; U.S. Patent Application Publication Nos. 2003/0068633 and2002/0081582; U.S. Pat. Nos. 6,833,450, 6,830,890, 6,824,866, 6,800,439,6,375,903 and 5,700,637; and PCT Application Nos. WO 04/031399, WO04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO 03/065038, WO03/064699, WO 03/064027, WO 03/064026, WO 03/046223, WO 03/040410 and WO02/24597.

Solid Phase Supports

In certain exemplary embodiments, one or more template nucleic acidsequences, i.e. oligonucleotide sequences, described herein areimmobilized on a support (e.g., a solid and/or semi-solid support). Incertain aspects, an oligonucleotide sequence can be attached to asupport using one or more of the phosphoramidite linkers describedherein. Suitable supports include, but are not limited to, slides,beads, chips, particles, strands, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates and the like. Invarious embodiments, a solid support may be biological, nonbiological,organic, inorganic, or any combination thereof. When using a supportthat is substantially planar, the support may be physically separatedinto regions, for example, with trenches, grooves, wells, or chemicalbarriers (e.g., hydrophobic coatings, etc.).

In certain exemplary embodiments, a support is a microarray. As usedherein, the term “microarray” refers in one embodiment to a type ofassay that comprises a solid phase support having a substantially planarsurface on which there is an array of spatially defined non-overlappingregions or sites that each contain an immobilized hybridization probe.“Substantially planar” means that features or objects of interest, suchas probe sites, on a surface may occupy a volume that extends above orbelow a surface and whose dimensions are small relative to thedimensions of the surface. For example, beads disposed on the face of afiber optic bundle create a substantially planar surface of probe sites,or oligonucleotides disposed or synthesized on a porous planar substratecreates a substantially planar surface. Spatially defined sites mayadditionally be “addressable” in that its location and the identity ofthe immobilized probe at that location are known or determinable.

Oligonucleotides immobilized on microarrays include nucleic acids thatare generated in or from an assay reaction. Typically, theoligonucleotides or polynucleotides on microarrays are single strandedand are covalently attached to the solid phase support, usually by a5′-end or a 3′-end. In certain exemplary embodiments, probes areimmobilized via one or more of the cleavable linkers described herein.The density of non-overlapping regions containing nucleic acids in amicroarray is typically greater than 100 per cm², and more typically,greater than 1000 per cm². Microarray technology relating to nucleicacid probes is reviewed in the following exemplary references: Schena,Editor, Microarrays: A Practical Approach (IRL Press, Oxford, 2000);Southern, Current Opin. Chem. Biol., 2: 404-410 (1998); Nature GeneticsSupplement, 21:1-60 (1999); and Fodor et al, U.S. Pat. Nos. 5,424,186;5,445,934; and 5,744,305. Methods of immobilizing oligonucleotides to asupport are known in the art (beads: Dressman et al. (2003) Proc. Natl.Acad. Sci. USA 100:8817, Brenner et al. (2000) Nat. Biotech. 18:630,Albretsen et al. (1990) Anal. Biochem. 189:40, and Lang et al. NucleicAcids Res. (1988) 16:10861; nitrocellulose: Ranki et al. (1983) Gene21:77; cellulose: Goldkorn (1986) Nucleic Acids Res. 14:9171;polystyrene: Ruth et al. (1987) Conference of Therapeutic and DiagnosticApplications of Synthetic Nucleic Acids, Cambridge U.K.;teflon-acrylamide: Duncan et al. (1988) Anal. Biochem. 169:104;polypropylene: Polsky-Cynkin et al. (1985) Clin. Chem. 31:1438; nylon:Van Ness et al. (1991) Nucleic Acids Res. 19:3345; agarose:Polsky-Cynkin et al., Clin. Chem. (1985) 31:1438; and sephacryl:Langdale et al. (1985) Gene 36:201; latex: Wolf et al. (1987) NucleicAcids Res. 15:2911).

As used herein, the term “attach” refers to both covalent interactionsand noncovalent interactions. A covalent interaction is a chemicallinkage between two atoms or radicals formed by the sharing of a pair ofelectrons (i.e., a single bond), two pairs of electrons (i.e., a doublebond) or three pairs of electrons (i.e., a triple bond). Covalentinteractions are also known in the art as electron pair interactions orelectron pair bonds. Noncovalent interactions include, but are notlimited to, van der Waals interactions, hydrogen bonds, weak chemicalbonds (i.e., via short-range noncovalent forces), hydrophobicinteractions, ionic bonds and the like. A review of noncovalentinteractions can be found in Alberts et al., in Molecular Biology of theCell, 3d edition, Garland Publishing, 1994.

Sequencing Primers

Sequencing primers according to the present disclosure are those thatare capable of binding to a known binding region of the targetpolynucleotide and facilitating ligation of an oligonucleotide probe ofthe present disclosure. Sequencing primers may be designed with the aidof a computer program such as, for example, DNAWorks, or Gene2Oligo. Thebinding region can vary in length but it should be long enough tohybridize the sequencing primer. Target polynucleotides may havemultiple different binding regions thereby allowing different sectionsof the target polynucleotide to be sequenced. Sequencing primers areselected to form highly stable duplexes so that they remain hybridizedduring successive cycles of ligation. Sequencing primers can be selectedsuch that ligation can proceed in either the 5′ to 3′ direction or the3′ to 5′ direction or both. Sequencing primers may contain modifiednucleotides or bonds to enhance their hybridization efficiency, orimprove their stability, or prevent extension from a one terminus or theother.

According to one aspect, single stranded DNA templates (ssDNA) areprepared by RCA as described above to be used with sequencing primers.Alternatively single stranded template is attached to beads ornanoparticles in an emulsion and amplified through ePCR. The result isclonal beads with a single amplified ssDNA template.

For the purpose of identifying several template nucleotide sequences inparallel, the templates are diluted in PBS buffer pH 7.4, and eitherbound to a patterned or non-patterned substrate utilizing variousattachment methods, such as Biotin-Strepavidin, azide-alkyle (e.g. clickchemistry), NHS-ester or Silanization (e.g. aldehyde-, epoxy-,amino-silane). According to one aspect, rolonies are attached to apatterned surface, such as a SiO₂ solid surface, treated with 1%aminosilane (v/v) and let to interact for a period of time (typicallybetween 5 minutes to 2 hours). Any unbound templates are then washedaway using Wash 1 buffer.

Next, a sequencing primer is prepared and hybridized to the sequencingprimer hybridizing site. According to certain aspects, sequencingprimers can be prepared which can hybridize to a known sequence of thetemplate. Alternatively, during template preparation, adapters with aknown nucleic acid sequence are added to the unknown nucleic acidsequence by way of ligation, amplification, transposition orrecombination according to methods known to those of skill in the artand described herein. Still alternatively, sequencing primers having acertain level of degeneracy could be used to hybridize to certainpositions along the template. According to one aspect, primer degeneracyis used to allow primers to hybridize semi-randomly along the template.Primer degeneracy is selected based on statistical methods known tothose of skill in the art to facilitate primers hybridizing at certainintervals along the length of the template. According to this aspect,primers can be designed having a certain degeneracy which facilitatesbinding every N bases, such as every 100 bases, every 200 bases, every2000 bases, every 100,000 bases. The binding of the primers along thelength of the template is based on the design of the primers and thestatistical likelihood that a primer design will bind about every Nbases along the length of the template. Since the sequencing primer P1will be extended by ligation, the terminal group of the sequencingprimer is typically synthesized to be ready to be covalently joined tothe oligonucleotide probe by the DNA ligase. If the ligation occursbetween the 5′ end of the sequencing primer and the 3′ end of theoligonucleotide probe, a phosphate group (5′-PO4) must be present on thesequencing primer while a hydroxyl group (3′-OH) on the oligonucleotideprobe, and vice-versa. To hybridize the sequencing primer to thesequencing primer hybridizing site, 1 uM of the sequencing primerdiluted in 5×SSPE buffer is used. The mixture is then incubated for afew minutes above room temperature to encourage proper annealing(typically between 1 to 5 minutes, at temperature between 25 and 55°C.).

Oligonucleotide Probes

Oligonucleotide probes according to the present disclosure are thosehaving between about 1 nucleotide to about 100 nucleotides. Exemplaryoligonucleotide probes include between about 1 nucleotide to about 20nucleotides, between about 3 nucleotides to about 15 nucleotides,between about 5 nucleotides to about 12 nucleotides or between about 6nucleotides to about 10 nucleotides. An exemplary oligonucleotide probeincludes about 6 nucleotides. According to one aspect, oligonucleotideprobes according to the present disclosure should be capable ofhybridizing to the single stranded nucleic acid template. According toan additional aspect, oligonucleotide probes according to the presentdisclosure may be capable of hybridizing to the single stranded nucleicacid template and ligating to a sequencing primer or an extended duplexto generate the extended duplex for the next ligation cycle. Accordingto a still additional aspect, a combination of oligonucleotide probescan be used where a first probe is capable of hybridizing to the singlestranded nucleic acid template and ligating to a sequencing primer or anextended duplex to generate the extended duplex for the next ligationcycle and a second probe is capable of hybridizing to the singlestranded nucleic acid template. Probes according to the presentdisclosure may include a nucleic acid origami structure. Oligonucleotideprobes may be designed with the aid of a computer program such as, forexample, DNAWorks, or Gene2Oligo.

Oligonucleotide probes according to the present disclosure may include aterminal moiety which prevents multiple ligations in a single ligationcycle. Oligonucleotide probes according to the present disclosure shouldalso be capable of being modified to create or include an extendableterminus for further ligation if an extendable terminus is not alreadypresent. Oligonucleotide probes according to the present disclosure neednot form a perfectly matched duplex with the single stranded nucleicacid template, though a perfect matched duplex is exemplary. Instead,oligonucleotide probes need only have a perfect match between anucleotide in the probe and the complementary nucleotide beingidentified by the methods described herein.

Hybridization and Ligation of Oligonucleotide Probes

Methods of hybridizing and ligating oligonucleotide probes to a singlestranded template nucleic acid are known to those of skill in the art.“Hybridization” refers to the process in which two single-strandedpolynucleotides bind non-covalently to form a stable double-strandedpolynucleotide. The term “hybridization” may also refer totriple-stranded hybridization. The resulting (usually) double-strandedpolynucleotide is a “hybrid” or “duplex.” “Hybridization conditions”will typically include salt concentrations of less than about 1 M, moreusually less than about 500 mM and even more usually less than about 200mM. Hybridization temperatures can be as low as 5° C., but are typicallygreater than 22° C., more typically greater than about 30° C., and oftenin excess of about 37° C. Hybridizations are usually performed understringent conditions, i.e., conditions under which a probe willhybridize to its target subsequence. Stringent conditions aresequence-dependent and are different in different circumstances. Longerfragments may require higher hybridization temperatures for specifichybridization. As other factors may affect the stringency ofhybridization, including base composition and length of thecomplementary strands, presence of organic solvents and extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one alone. Generally, stringent conditions areselected to be about 5° C. lower than the T_(n), for the specificsequence at s defined ionic strength and pH. Exemplary stringentconditions include salt concentration of at least 0.01 M to no more than1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and atemperature of at least 25° C. For example, conditions of 5×SSPE (750 mMNaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C. are suitable for allele-specific probe hybridizations. For stringentconditions, see for example, Sambrook, Fritsche and Maniatis, MolecularCloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989) andAnderson Nucleic Acid Hybridization, 1^(st) Ed., BIOS ScientificPublishers Limited (1999). “Hybridizing specifically to” or“specifically hybridizing to” or like expressions refer to the binding,duplexing, or hybridizing of a molecule substantially to or only to aparticular nucleotide sequence or sequences under stringent conditionswhen that sequence is present in a complex mixture (e.g., totalcellular) DNA or RNA.

Ligation can be accomplished either enzymatically or chemically.“Ligation” means to form a covalent bond or linkage between the terminiof two or more nucleic acids, e.g., oligonucleotides and/orpolynucleotides, in a template-driven reaction. The nature of the bondor linkage may vary widely and the ligation may be carried outenzymatically or chemically. As used herein, ligations are usuallycarried out enzymatically to form a phosphodiester linkage between a 5′carbon of a terminal nucleotide of one oligonucleotide with 3′ carbon ofanother oligonucleotide. A variety of template-driven ligation reactionsare described in the following references: Whitely et al., U.S. Pat. No.4,883,750; Letsinger et al., U.S. Pat. No. 5,476,930; Fung et al., U.S.Pat. No. 5,593,826; Kool, U.S. Pat. No. 5,426,180; Landegren et al.,U.S. Pat. No. 5,871,921; Xu and Kool (1999) Nucl. Acids Res. 27:875;Higgins et al., Meth. in Enzymol. (1979) 68:50; Engler et al. (1982) TheEnzymes, 15:3 (1982); and Namsaraev, U.S. Patent Pub. 2004/0110213.

Chemical ligation methods are disclosed in Ferris et al., Nucleosides &Nucleotides, 8: 407-414 (1989) and Shabarova et al., Nucleic Acidsresearch, 19: 4247-4251 (1991). Enzymatic ligation utilizes a ligase.Many ligases are known to those of skill in the art as referenced inLehman, Science, 186: 790-797 (1974); Engler et al., DNA ligases, pages3-30 in Boyer, editor, The Enzymes, Vol. 15B (Academic Press, New York,1982); and the like. Exemplary ligases include T4 DNA ligase, T7 DNAligase, E. coli DNA ligase, Taq ligase, Pfu ligase and the like. Certainprotocols for using ligases are disclosed by the manufacturer and alsoin Sambrook, Molecular Cloning: A Laboratory manual, 2^(nd) Edition(Cold Spring Harbor Laboratory, New York, 1989); barany, PCR Methods andApplications, 1:5-16 (1991); Marsh et al., Strategies, 5:73-76 (1992).

If ligation is not 100% efficient, it may be desirable to cap extendedduplexes that fail to undergo ligation so that they do not participatein further ligation steps. According to certain aspects, capping can bedone by removing the 5′phosphate (5′PO) using an alkaline phosphatase.By example, following ligation of the oligonucleotide probes forsequencing, unreacted 5′PO are removed by adding an alkaline phosphatasein solution, such as 10 units of calf insetting alkaline phosphatase(NEB # M0393L) in 100 μL of its reaction buffer. The reaction isincubated for 15 minutes at room temperature. Other alkalinephosphatases are suitable. Capping can also be done by using apolymerase, deficient in exonuclease activity, to add a terminalnucleotide in the 5′->3′ direction (so capping the 3′ end of a primer).Terminal nucleotide varies but most frequently used aredideoxynucleotides (ddNTP) and acyclonucleotides (acyNTP). Anontemplated nucleotide can also be used as a terminal nucleotide.Capping by polymerase extension is performed as described to amplify apolynucleotide sequence using DNA polymerases, except that dNTP normallyused in the reaction are substituted by terminal NTP (e.g. ddNTP), whichprevent the DNA polymerase or Terminal Transferase (TdT) of adding morethan one nucleotide. By example, following ligation of theoligonucleotide probes for sequencing, a capping mix is added, whichconsists of 1 mM of ddNTP and 20 units of Terminal Transferase (NEB #M0315L) in 100 μL of its reaction buffer. The reaction is incubated for15 minutes at room temperature. Alternatively, capping can be done byligating an oligonucleotide, ideally between 6-9mer long, with a cappedend. The cap can be in the form of 5′hydroxyl (5′OH), instead of 5′PO,and oppositely 3′PO instead of 3′OH, a terminal NTP (ddNTP, invertedddNTP, acyNTP) or an oligo with a terminal carbon spacer (e.g. C3spacer). This method would work as well for capping the 5′end or the3′end of the polynucleotyde sequence to be capped. Capping by ligationis performed as described for ligating an oligonucleotide probe. Byexample, following ligation of the oligonucleotide probes forsequencing, a capping mix is added, which consists of 1 uM of a 5′- or3′-capped oligonucleotide added to the ligation buffer with 1200 unitsof T4 DNA ligase, per 100 μL reaction volume. The reaction is incubatedfor 15 minutes at room temperature.

According to the present disclosure, a specific set of oligonucleotideprobes L1 is utilized to hybridize to the ssDNA template and covalentlylinked to the sequencing primer P1 by a DNA ligase. Oligonucleotideprobes L1 are prepared in ligation buffer (typically at 1 uM), andligated using 6000 units of T3 DNA ligase (Enzymatics # L601L) or 1200units of T4 DNA ligase (Enzymatics # L603-HC-L) per 100 μL reactionvolume. The reaction is allowed to incubate at room temperature for afew minutes to several hours (typically between 5 minutes to 2 hours, ata temperature between 15° C. and 35° C.). Then the enzymes and anyunligated oligonucleotide probes L1 are washed away with wash 1 buffer.

Hybridization Conditions

In certain exemplary embodiments, the terms “annealing” and“hybridization,” as used herein, are used interchangeably to mean theformation of a stable duplex. In one aspect, stable duplex means that aduplex structure is not destroyed by a stringent wash under conditionssuch as a temperature of either about 5° C. below or about 5° C. abovethe T_(m) of a strand of the duplex and low monovalent saltconcentration, e.g., less than 0.2 M, or less than 0.1 M or saltconcentrations known to those of skill in the art. The term “perfectlymatched,” when used in reference to a duplex means that thepolynucleotide and/or oligonucleotide strands making up the duplex forma double stranded structure with one another such that every nucleotidein each strand undergoes Watson-Crick base pairing with a nucleotide inthe other strand. The term “duplex” includes, but is not limited to, thepairing of nucleoside analogs, such as deoxyinosine, nucleosides with2-aminopurine bases, PNAs, and the like, that may be employed. A“mismatch” in a duplex between two oligonucleotides means that a pair ofnucleotides in the duplex fails to undergo Watson-Crick bonding.

As used herein, the term “hybridization conditions,” will typicallyinclude salt concentrations of less than about 1 M, more usually lessthan about 500 mM and even more usually less than about 200 mM.Hybridization temperatures can be as low as 5° C., but are typicallygreater than 22° C., more typically greater than about 30° C., and oftenin excess of about 37° C. Hybridizations are usually performed understringent conditions, e.g., conditions under which a probe willspecifically hybridize to its target subsequence. Stringent conditionsare sequence-dependent and are different in different circumstances.Longer fragments may require higher hybridization temperatures forspecific hybridization. As other factors may affect the stringency ofhybridization, including base composition and length of thecomplementary strands, presence of organic solvents and extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one alone.

Generally, stringent conditions are selected to be about 5° C. lowerthan the T_(m) for the specific sequence at a defined ionic strength andpH. Exemplary stringent conditions include salt concentration of atleast 0.01 M to no more than 1 M Na ion concentration (or other salts)at a pH 7.0 to 8.3 and a temperature of at least 25° C. For example,conditions of 5×SSPE (750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH7.4) and a temperature of 25-30° C. are suitable for allele-specificprobe hybridizations. For stringent conditions, see for example,Sambrook, Fritsche and Maniatis, Molecular Cloning A Laboratory Manual,2nd Ed. Cold Spring Harbor Press (1989) and Anderson Nucleic AcidHybridization, 1^(st) Ed., BIOS Scientific Publishers Limited (1999). Asused herein, the terms “hybridizing specifically to” or “specificallyhybridizing to” or similar terms refer to the binding, duplexing, orhybridizing of a molecule substantially to a particular nucleotidesequence or sequences under stringent conditions.

Nucleic Acid Origami Structures

According to certain aspects of the present disclosure, a nucleic acidorigami structure is a two dimensional structure or a three dimensionalstructure which is created from DNA. The terms spatially distinctnucleic acid structure, geometrically distinct nucleic acid structure,spatially resolvable nucleic acid structure, spatially observablenucleic acid structure are intended to include the term DNA origami. DNAorigami may be a megadalton-scale DNA nanostructure created from one ormore or a plurality of DNA strands. According to an exemplary aspect, anucleic acid origami structure is created from a scaffold strand of anucleic acid, such as DNA, which is arranged into a desiredmacromolecular object of a custom shape. Staples strands of DNA, whichmay be shorter than the scaffold strand of DNA, can be used to directthe folding or other orientation of the scaffold strand of DNA into aprogrammed arrangement. The term “origami” infers that one or morestrands or building blocks of DNA may be folded or otherwise positionedinto a desired structure or shape. The desired structure or shape whichmay then be secured into a desired shape or structure by one or moreother strands or building blocks of DNA, such as a plurality of staplestrands of DNA. Methods of making DNA origami are known to those ofskill in the art. Representative methods include Rothemund, “Folding DNAto Create Nanoscale Shapes and Patterns”, Nature March 2006, p. 297-302,vol. 440; Rothemund, “Design of DNA Origami”, Proceedings of theInternational Conference of Computer-Aided Design (ICCAD) 2005; U.S.Pat. No. 7,842,793; Douglas et al., Nuc. Acids Res., vol. 37, no. 15,pp. 5001-5006; and Douglas et al., Nature, 459, pp. 414-418 (2009);Andersen et al., Nature, 459, pp. 73-76 (2009); Deitz et al., Science,325, pp. 725-730 (2009); Han et al., Science, 332, pp. 342-346 (2011);Liu et al., Angew. Chem. Int. Ed., 50, pp. 264-267 (2011); Zhao et al.,Nano Lett., 11, pp. 2997-3002 (2011); Woo et al., Nat. Chem. 3, pp.620-627 (2011) Torring et al., Chem. Soc. Rev. 40, pp. 5636-5646 (2011)each of which are hereby incorporated by reference in their entireties.According to an exemplary aspect, a nucleic acid origami structure neednot be constructed of a scaffold strand and staple strands. A nucleicacid origami structure can be constructed by single stranded nucleicacid sequences which self-assemble into tiles to form lattices of anydesired shape or size. Such single stranded nucleic acid sequences maybe de novo designed and synthesized. Such approaches include programmedself-assembly of such designed strands of nucleic acids to create a widerange of structures with desired shapes. See Wei et al., Nature, volume485, pp. 623-627 (2012) hereby incorporated by reference in itsentirety.

It is to be understood that the principles of the present disclosure donot rely on any particular method of making DNA origami or anyparticular two dimensional or three dimensional nucleic acid shape. Itis to be understood that aspects of the ability of DNA origami toprovide unique shapes is useful to barcode or otherwise identifyspecific nucleic acids or nucleic acid sequences. It is to be furtherunderstood that aspects of the ability to design DNA origami withdesired hybridization sites or desired probes is useful to barcode orotherwise identify specific nucleic acids or nucleic acid sequences. Itis to be further understood that the ability of DNA origami to be ofsufficient size to be identified by visualizing the shape of the DNAorigami is useful to barcode or otherwise identify specific nucleicacids or nucleic acid sequences. It is to be further understood that theability of DNA origami to be of sufficient size to be directly visuallydistinguishable is useful to barcode or otherwise identify specificnucleic acids or nucleic acid sequences. It is to be further understoodthat the ability of DNA origami to be megadalton-scale nucleic acid(such as DNA) nanostructures of sufficient size to be identified byvisualizing the shape of the DNA origami is useful to barcode orotherwise identify specific nucleic acids or nucleic acid sequences.According to certain aspects, the DNA origami are visuallydistinguishable or identifiable without the aid of fluorescentdetectable moieties. According to certain aspects, a set of DNA origamiare provided which are each distinct based on the shape of the DNAorigami or the presence and/or position of detectable moieties on theDNA origami. According to certain aspects, a set of DNA origami barcodesare provided which are used to uniquely identify a nucleic acid ornucleic acid sequence such as a nucleic acid probe.

According to certain aspects of the present disclosure, a nucleic acidorigami structure is attached to an oligonucleotide probe. The nucleicacid origami structure may include a detectable moiety, label, reporterand/or barcode. The nucleic acid origami structure may include a probehybridization site for hybridizing with a probe having a detectablemoiety, label, reporter or barcode. The nucleic acid origami structuremay have a geometrically distinct or geometrically unique structure.

Methods of making nucleic acid origami structures are known to those ofskill in the art. Methods of attaching a detectable moiety, label orreporter to a nucleic acid sequence are known to those of skill in theart.

Detectable Moieties

In certain exemplary embodiments, a detectable moiety, label or reportercan be used to detect one or more nucleotides described herein.Oligonucleotides described herein can be labeled in a variety of ways,including the direct or indirect attachment of a detectable moiety suchas a fluorescent moiety, colorimetric moiety and the like. One of skillin the art can consult references directed to labeling DNA. Examples ofdetectable moieties include various radioactive moieties, enzymes,prosthetic groups, fluorescent markers, luminescent markers,bioluminescent markers, metal particles, protein-protein binding pairs,protein-antibody binding pairs and the like. Examples of fluorescentmoieties include, but are not limited to, yellow fluorescent protein(YFP), green fluorescence protein (GFP), cyan fluorescence protein(CFP), umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansylchloride, phycocyanin, phycoerythrin and the like. Examples ofbioluminescent markers include, but are not limited to, luciferase(e.g., bacterial, firefly, click beetle and the like), luciferin,aequorin and the like. Examples of enzyme systems having visuallydetectable signals include, but are not limited to, galactosidases,glucorinidases, phosphatases, peroxidases, cholinesterases and the like.Identifiable markers also include radioactive compounds such as ¹²⁵I,³⁵S, ¹⁴C, or ³H. Identifiable markers are commercially available from avariety of sources.

Fluorescent labels and their attachment to nucleotides and/oroligonucleotides are described in many reviews, including Haugland,Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition(Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes,2nd Edition (Stockton Press, New York, 1993); Eckstein, editor,Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford,1991); and Wetmur, Critical Reviews in Biochemistry and MolecularBiology, 26:227-259 (1991). Particular methodologies applicable to theinvention are disclosed in the following sample of references: U.S. Pat.Nos. 4,757,141, 5,151,507 and 5,091,519. In one aspect, one or morefluorescent dyes are used as labels for labeled target sequences, e.g.,as disclosed by U.S. Pat. No. 5,188,934 (4,7-dichlorofluorescein dyes);U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes); U.S.Pat. No. 5,847,162 (4,7-dichlororhodamine dyes); U.S. Pat. No. 4,318,846(ether-substituted fluorescein dyes); U.S. Pat. No. 5,800,996 (energytransfer dyes); Lee et al.; U.S. Pat. No. 5,066,580 (xanthine dyes);U.S. Pat. No. 5,688,648 (energy transfer dyes); and the like. Labellingcan also be carried out with quantum dots, as disclosed in the followingpatents and patent publications: U.S. Pat. Nos. 6,322,901, 6,576,291,6,423,551, 6,251,303, 6,319,426, 6,426,513, 6,444,143, 5,990,479,6,207,392, 2002/0045045 and 2003/0017264. As used herein, the term“fluorescent label” includes a signaling moiety that conveys informationthrough the fluorescent absorption and/or emission properties of one ormore molecules. Such fluorescent properties include fluorescenceintensity, fluorescence lifetime, emission spectrum characteristics,energy transfer, and the like.

Commercially available fluorescent nucleotide analogues readilyincorporated into nucleotide and/or oligonucleotide sequences include,but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (AmershamBiosciences, Piscataway, N.J.), fluorescein-12-dUTP,tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP,BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINEGREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY™630/650-14-dUTP, BODIPY™ 650/665-14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXAFLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP,ALEXA FLUOR™ 546-14-dUTP, fluorescein-12-UTP,tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADEBLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR-14-UTP, BODIPY™ TR-14-UTP,RHODAMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, LEXA FLUOR™ 546-14-UTP(Molecular Probes, Inc. Eugene, Oreg.) and the like. Alternatively, theabove fluorophores and those mentioned herein may be added duringoligonucleotide synthesis using for example phosphoroamidite or NHSchemistry. Protocols are known in the art for custom synthesis ofnucleotides having other fluorophores (See, Henegariu et al. (2000)Nature Biotechnol. 18:345). 2-Aminopurine is a fluorescent base that canbe incorporated directly in the oligonucleotide sequence during itssynthesis. Nucleic acid could also be stained, a priori, with anintercalating dye such as DAPI, YOYO-1, ethidium bromide, cyanine dyes(e.g. SYBR Green) and the like.

Other fluorophores available for post-synthetic attachment include, butare not limited to, ALEXA FLUOR™ 350, ALEXA FLUOR™ 405, ALEXA FLUOR™430, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™594, ALEXA FLUOR™ 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570,BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B,Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, PacificOrange, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene,Oreg.), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 (Amersham Biosciences,Piscataway, N.J.) and the like. FRET tandem fluorophores may also beused, including, but not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5,PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexadyes and the like.

Metallic silver or gold particles may be used to enhance signal fromfluorescently labeled nucleotide and/or oligonucleotide sequences(Lakowicz et al. (2003) BioTechniques 34:62).

Biotin, or a derivative thereof, may also be used as a label on anucleotide and/or an oligonucleotide sequence, and subsequently bound bya detectably labeled avidin/streptavidin derivative (e.g.phycoerythrin-conjugated streptavidin), or a detectably labeledanti-biotin antibody. Digoxigenin may be incorporated as a label andsubsequently bound by a detectably labeled anti-digoxigenin antibody(e.g. fluoresceinated anti-digoxigenin). An aminoallyl-dUTP oraminohexylacrylamide-dCTP residue may be incorporated into anoligonucleotide sequence and subsequently coupled to an N-hydroxysuccinimide (NHS) derivatized fluorescent dye. In general, any member ofa conjugate pair may be incorporated into a detection oligonucleotideprovided that a detectably labeled conjugate partner can be bound topermit detection. As used herein, the term antibody refers to anantibody molecule of any class, or any sub-fragment thereof, such as anFab.

Other suitable labels for an oligonucleotide sequence may includefluorescein (FAM, FITC), digoxigenin, dinitrophenol (DNP), dansyl,biotin, bromodeoxyuridine (BrdU), hexahistidine (6×His), phosphor-aminoacids (e.g. P-tyr, P-ser, P-thr) and the like. In one embodiment thefollowing hapten/antibody pairs are used for detection, in which each ofthe antibodies is derivatized with a detectable label: biotin/α-biotin,digoxigenin/α-digoxigenin, dinitrophenol (DNP)/α-DNP,5-Carboxyfluorescein (FAM)/α-FAM.

In certain exemplary embodiments, a nucleotide and/or an oligonucleotidesequence can be indirectly labeled, especially with a hapten that isthen bound by a capture agent, e.g., as disclosed in U.S. Pat. Nos.5,344,757, 5,702,888, 5,354,657, 5,198,537 and 4,849,336, PCTpublication WO 91/17160 and the like. Many different hapten-captureagent pairs are available for use. Exemplary haptens include, but arenot limited to, biotin, des-biotin and other derivatives, dinitrophenol,dansyl, fluorescein, CY5, digoxigenin and the like. For biotin, acapture agent may be avidin, streptavidin, or antibodies. Antibodies maybe used as capture agents for the other haptens (many dye-antibody pairsbeing commercially available, e.g., Molecular Probes, Eugene, Oreg.).

According to certain aspects, detectable moieties described herein arespectrally resolvable. “Spectrally resolvable” in reference to aplurality of fluorescent labels means that the fluorescent emissionbands of the labels are sufficiently distinct, i.e., sufficientlynon-overlapping, that molecular tags to which the respective labels areattached can be distinguished on the basis of the fluorescent signalgenerated by the respective labels by standard photodetection systems,e.g., employing a system of band pass filters and photomultiplier tubes,or the like, as exemplified by the systems described in U.S. Pat. Nos.4,230,558; 4,811,218, or the like, or in Wheeless et al., pgs. 21-76, inFlow Cytometry: Instrumentation and Data Analysis (Academic Press, NewYork, 1985). In one aspect, spectrally resolvable organic dyes, such asfluorescein, rhodamine, and the like, means that wavelength emissionmaxima are spaced at least 20 nm apart, and in another aspect, at least40 nm apart. In another aspect, chelated lanthanide compounds, quantumdots, and the like, spectrally resolvable means that wavelength emissionmaxima are spaced at least 10 nm apart, and in a further aspect, atleast 15 nm apart.

In certain embodiments, the detectable moieties can provide higherdetectability when used with an electron microscope, compared withcommon nucleic acids. Moieties with higher detectability are often inthe group of metals and organometals, such as mercuric acetate, platinumdimethylsulfoxide, several metal-bipyridyl complexes (e.g. osmium-bipy,ruthenium-bipy, platinum-bipy). While some of these moieties can readilystain nucleic acids specifically, linkers can also be used to attachthese moieties to a nucleic acid. Such linkers added to nucleotidesduring synthesis are acrydite- and a thiol-modified entities, aminereactive groups, and azide and alkyne groups for performing clickchemistry. Some nucleic acid analogs are also more detectable such asgamma-adenosine-thiotriphosphate, iododeoxycytidine-triphosphate, andmatellonucleosides in general (see Dale et al., Proc. Nat. Acad. Sci.USA, Vol. 70, No. 8, pp. 2238-2242 (1973)). The modified nucleotides areadded during synthesis. Synthesis may refer by example to solid supportsynthesis of oligonucleotides. In this case, modified nucleic acids,which can be a nucleic acid analog, or a nucleic acid modified with adetectable moiety, or with an attachment chemistry linker, are added oneafter each other to the nucleic acid fragments being formed on the solidsupport, with synthesis by phosphoramidite being the most popularmethod. Synthesis may also refer to the process performed by apolymerase while it synthesizes the complementary strands of a nucleicacid template. Certain DNA polymerases are capable of using andincorporating nucleic acids analogs, or modified nucleic acids, eithermodified with a detectable moiety or an attachment chemistry linker tothe complementary nucleic acid template.

Cleavable Moieties

According to certain aspects of the present disclosure, cleavablenucleotide moieties also referred to as cleavable linkages are used toseparate an oligonucleotide probe from a nucleic acid origami structure.Cleavable moieties are known to those of skill in the art and includechemically scissile internucleosidic linkages which may be cleaved bytreating them with chemicals or subjecting them to oxidizing or reducingenvironments. Such cleavable moieties include phosphorothioate,phosphorothiolate which can be cleaved by various metal ions such assolutions of silver nitrate. Such cleavable moieties includephosphoroamidate which can be cleaved in acidic conditions such assolutions including acetic acid. A suitable chemical that can cleave alinkage includes a chemical that can cleave a bridged-phosphorothioatelinkage and can remove a phosphoramidite linker from a nucleotide and/oroligonucleotide, leaving a free phosphate group on the nucleotide and/oroligonucleotide at the cleavage site. Suitable chemicals include, butare not limited to AgNO₃, AgCH₃COO, AgBrO₃, Ag₂SO₄, or any compound thatdelivers Ag²⁺, HgCl₂, I₂, Br₂, I⁻, Br⁻ and the like.

Cleavable moieties also include those that can be cleaved by nucleasesknown to those of skill in the art. Such nucleases include restrictionendonucleases such as Type I, Type II, Type III and Type IV,endonucleases such as endonucleases I-VIII, ribonucleases and othernucleases such as enzymes with AP endonuclease activity, enzymes with APlyase activity and enzymes with glycosylase activity such as uracil DNAglycosylase.

Cleavable moieties also include those capable of being cleaved by lightof a certain wavelength. Such cleavable moieties are referred to asphotolabile linkages and are disclosed in Olejnik et al., Photocleavablebiotin derivatives: a versatile approach for the isolation ofbiomolecules, Proc. Natl. Acad. Sci. U.S.A., vol. 92, p. 7590-7594(1995). Such photocleavable linkers can be cleaved by UV illuminationbetween wavelengths of about 275 to about 375 nm for a period of a fewseconds to 30 minutes, such as about one minute. Exemplary wavelengthsinclude between about 300 nm to about 350 nm.

Certain nucleotides, such as dGTP, dCTP and dTTP could also be reactedbefore being incorporated for use as a cleavable linkage, making themspecifically sensitive to further cleavage by nucleases or chemicals.According to one aspect, one or multiple deoxyguanosines in a giventemplate non-hybridizing nucleic acid can be oxidized to8-oxo-deoxyguanosine by 2-nitropropane, before being added to thesequencing reaction, and subsequently cleaved using an 8-oxoguanine DNAglycosylase (e.g. Fpg, hOGG1). Similarly, deoxycytosines can bepre-reacted to form 5-hydroxycytosine, using bisulfite or nitrous acid,which can then be processed by certain DNA-glycosylase, such as hNEIL1.Other nucleotides which can be cleaved include uracil, deoxyuridine,inosine and deoxyinosine.

Additional embodiments include nucleotides that may be cleaved in a twostep method such as by a first step that modifies the nucleotide makingit more susceptible to cleavage and then a second step where thenucleotide is cleaved. Such systems include the USER system(commercially available from Enzymatics (# Y918L) or New England Biolabs(# M5505L) which is typically a combination of UDG and EndonucleaseVIII, although other endonucleases could be used. Enzymes UDG andendonuclease are commercially available. In addition, modifiednucleotides may be cleavable nucleotides where a feature of thenucleotide has been modified, such as a bond, so as to facilitatecleavage. Examples include an abasic base, an apyrimidic base, anapurinic base, phospohrothioate, phosphorothiolate and oxidized basessuch as deoxyguanosines which can be oxidized to 8-oxo-deoxyguanosine.

Accordingly, internucleotide bonds may be cleaved by chemical, thermal,or light based cleavage. Exemplary chemically cleavable internucleotidelinkages for use in the methods described herein include, for example,□-cyano ether, 5′-deoxy-5′-aminocarbamate, 3′deoxy-3′-aminocarbamate,urea, 2′cyano-3′,5′-phosphodiester, 3′-(S)-phosphorothioate,5′-(S)-phosphorothioate, 3′-(N)-phosphoramidate, 5′-(N)-phosphoramidate,O-amino amide, vicinal diol, ribonucleoside insertion,2′-amino-3′,5′-phosphodiester, allylic sulfoxide, ester, silyl ether,dithioacetal, 5′-thio-furmal, □-hydroxy-methyl-phosphonic bisamide,acetal, 3′-thio-furmal, methylphosphonate and phosphotriester.Internucleoside silyl groups such as trialkylsilyl ether anddialkoxysilane are cleaved by treatment with fluoride ion.Base-cleavable sites include □-cyano ether, 5′-deoxy-5′-aminocarbamate,3′-deoxy-3′-aminocarbamate, urea, 2′-cyano-3′,5′-phosphodiester,2′-amino-3′,5′-phosphodiester, ester and ribose. Thio-containinginternucleotide bonds such as 3′-(S)-phosphorothioate and5′-(S)-phosphorothioate are cleaved by treatment with silver nitrate ormercuric chloride. Acid cleavable sites include 3′-(N)-phosphoramidate,5′-(N)-phosphoramidate, dithioacetal, acetal and phosphonic bisamide. An□-aminoamide internucleoside bond is cleavable by treatment withisothiocyanate, and titanium may be used to cleave a2′-amino-3′,5′-phosphodiester-O-ortho-benzyl internucleoside bond.Vicinal diol linkages are cleavable by treatment with periodate.Thermally cleavable groups include allylic sulfoxide and cyclohexenewhile photo-labile linkages include nitrobenzylether and thymidinedimer. Methods synthesizing and cleaving nucleic acids containingchemically cleavable, thermally cleavable, and photo-labile groups aredescribed for example, in U.S. Pat. No. 5,700,642.

Accordingly, internucleotide bonds may be cleaved using enzymaticcleavage. Nucleic acid sequences described herein may be designed toinclude a restriction endonuclease cleavage site. A nucleic acid may becontacted with a restriction endonuclease to result in cleavage. A widevariety of restriction endonucleases having specific binding and/orcleavage sites are commercially available, for example, from New EnglandBiolabs (Ipswich, Mass.). In various embodiments, restrictionendonucleases that produce 3′ overhangs, 5′ overhangs or blunt ends maybe used. When using a restriction endonuclease that produces anoverhang, an exonuclease (e.g., RecJ_(f), Exonuclease I, Exonuclease T,S₁ nuclease, P₁ nuclease, mung bean nuclease, CEL I nuclease, etc.) maybe used to produce blunt ends. In an exemplary embodiment, an orthogonalprimer/primer binding site that contains a binding and/or cleavage sitefor a type IIS restriction endonuclease may be used to remove thetemporary orthogonal primer binding site.

As used herein, the term “restriction endonuclease recognition site” isintended to include, but is not limited to, a particular nucleic acidsequence to which one or more restriction enzymes bind, resulting incleavage of a DNA molecule either at the restriction endonucleaserecognition sequence itself, or at a sequence distal to the restrictionendonuclease recognition sequence. Restriction enzymes include, but arenot limited to, type I enzymes, type II enzymes, type IIS enzymes, typeIII enzymes and type IV enzymes. The REBASE database provides acomprehensive database of information about restriction enzymes, DNAmethyltransferases and related proteins involved inrestriction-modification. It contains both published and unpublishedwork with information about restriction endonuclease recognition sitesand restriction endonuclease cleavage sites, isoschizomers, commercialavailability, crystal and sequence data (see Roberts et al. (2005) Nucl.Acids Res. 33:D230, incorporated herein by reference in its entirety forall purposes).

In certain aspects, primers of the present invention include one or morerestriction endonuclease recognition sites that enable type IIS enzymesto cleave the nucleic acid several base pairs 3′ to the restrictionendonuclease recognition sequence. As used herein, the term “type IIS”refers to a restriction enzyme that cuts at a site remote from itsrecognition sequence. Type IIS enzymes are known to cut at a distancesfrom their recognition sites ranging from 0 to 20 base pairs. Examplesof Type IIs endonucleases include, for example, enzymes that produce a3′ overhang, such as, for example, Bsr I, Bsm I, BstF5 I, BsrD I, Bts I,Mnl I, BciV I, Hph I, Mbo II, Eci I, Acu I, Bpm I, Mme I, BsaX I, Bcg I,Bae I, Bfi I, TspDT I, TspGW I, Taq II, Eco57 I, Eco57M I, Gsu I, Ppi I,and Psr I; enzymes that produce a 5′ overhang such as, for example, BsmAI, Ple I, Fau I, Sap I, BspM I, SfaN I, Hga I, Bvb I, Fok I, BceA I,BsmF I, Ksp632 I, Eco31 I, Esp3 I, Aar I; and enzymes that produce ablunt end, such as, for example, Mly I and Btr I. Type-IIs endonucleasesare commercially available and are well known in the art (New EnglandBiolabs, Beverly, Mass.). Information about the recognition sites, cutsites and conditions for digestion using type IIs endonucleases may befound, for example, on the Worldwide web atneb.com/nebecomm/enzymefindersearch bytypeIIs.asp). Restrictionendonuclease sequences and restriction enzymes are well known in the artand restriction enzymes are commercially available (New England Biolabs,Ipswich, Mass.).

According to certain aspects, the cleavable moiety may be within anoligonucleotide and may be introduced during in situ synthesis. A broadvariety of cleavable moieties are available in the art of solid phaseand microarray oligonucleotide synthesis (see e.g., Pon, R., MethodsMol. Biol. 20:465-496 (1993); Verma et al., Ann. Rev. Biochem. 67:99-134(1998); U.S. Pat. Nos. 5,739,386, 5,700,642 and 5,830,655; and U.S.Patent Publication Nos. 2003/0186226 and 2004/0106728).

The cleavable site may be located along the oligonucleotide backbone,for example, a modified 3′-5′ internucleotide linkage in place of one ofthe phosphodiester groups, such as ribose, dialkoxysilane,phosphorothioate, and phosphoramidate internucleotide linkage. Thecleavable oligonucleotide analogs may also include a substituent on, orreplacement of, one of the bases or sugars, such as 7-deazaguanosine,5-methylcytosine, inosine, uridine, and the like.

In one embodiment, cleavable sites contained within the modifiedoligonucleotide may include chemically cleavable groups, such asdialkoxysilane, 3′-(S)-phosphorothioate, 5′-(S)-phosphorothioate,3′-(N)-phosphoramidate, 5′-(N)phosphoramidate, and ribose. Synthesis andcleavage conditions of chemically cleavable oligonucleotides aredescribed in U.S. Pat. Nos. 5,700,642 and 5,830,655. For example,depending upon the choice of cleavable site to be introduced, either afunctionalized nucleoside or a modified nucleoside dimer may be firstprepared, and then selectively introduced into a growing oligonucleotidefragment during the course of oligonucleotide synthesis. Selectivecleavage of the dialkoxysilane may be effected by treatment withfluoride ion. Phosphorothioate internucleotide linkage may beselectively cleaved under mild oxidative conditions. Selective cleavageof the phosphoramidate bond may be carried out under mild acidconditions, such as 80% acetic acid. Selective cleavage of ribose may becarried out by treatment with dilute ammonium hydroxide.

In another embodiment, a non-cleavable hydroxyl linker may be convertedinto a cleavable linker by coupling a special phosphoramidite to thehydroxyl group prior to the phosphoramidite or H-phosphonateoligonucleotide synthesis as described in U.S. Patent ApplicationPublication No. 2003/0186226. The cleavage of the chemicalphosphorylation agent at the completion of the oligonucleotide synthesisyields an oligonucleotide bearing a phosphate group at the 3′ end. The3′-phosphate end may be converted to a 3′ hydroxyl end by a treatmentwith a chemical or an enzyme, such as alkaline phosphatase, which isroutinely carried out by those skilled in the art.

In another embodiment, the cleavable linking moiety may be a TOPS (twooligonucleotides per synthesis) linker (see e.g., PCT publication WO93/20092). For example, the TOPS phosphoramidite may be used to converta non-cleavable hydroxyl group on the solid support to a cleavablelinker. A preferred embodiment of TOPS reagents is the Universal TOPS™phosphoramidite. Conditions for Universal TOPS phosphoramiditepreparation, coupling and cleavage are detailed, for example, in Hardyet al. Nucleic Acids Research 22(15):2998-3004 (1994). The UniversalTOPS phosphoramidite yields a cyclic 3′ phosphate that may be removedunder basic conditions, such as the extended ammonia and/orammonia/methylamine treatment, resulting in the natural 3′ hydroxyoligonucleotide.

In another embodiment, a cleavable linking moiety may be an aminolinker. The resulting oligonucleotides bound to the linker via aphosphoramidite linkage may be cleaved with 80% acetic acid yielding a3′-phosphorylated oligonucleotide.

In another embodiment, the cleavable linking moiety may be aphotocleavable linker, such as an ortho-nitrobenzyl photocleavablelinker. Synthesis and cleavage conditions of photolabileoligonucleotides on solid supports are described, for example, inVenkatesan et al., J. Org. Chem. 61:525-529 (1996), Kahl et al., J. Org.Chem. 64:507-510 (1999), Kahl et al., J. Org. Chem. 63:4870-4871 (1998),Greenberg et al., J. Org. Chem. 59:746-753 (1994), Holmes et al., J.Org. Chem. 62:2370-2380 (1997), and U.S. Pat. No. 5,739,386.Ortho-nitrobenzyl-based linkers, such as hydroxymethyl, hydroxyethyl,and Fmoc-aminoethyl carboxylic acid linkers, may also be obtainedcommercially.

Example I

Total human genomic DNA was extracted from a fibroblast cell cultureoriginally from a human skin biopsy, following methods to extractmillion bases long DNA by Pulse-Field Gel Electrophoresis (PFGE) anddescribed by Zhang et al., Nature Protocols, vol. 7, no. 3, pp. 467-478(2012), Michalet et al., Science, vol. 277, 1518 (1997) or CHEF GenomicDNA Plug Kits (Bio-Rad).

Fibroblast cells were grown to confluence (˜2×10⁶ cells/ml) usingstandard conditions, i.e. using complete growth medium DMEM (LifeTechnologies) containing 10% Fetal Bovine Serum (FBS), in an incubatorat 37° C. and 5% CO₂. Upon confluence, DMEM and FBS were removed bywashing with cold Phosphate Buffered Saline solution (PBS), and 0.05%trypsin-EDTA-PBS solution was added to the culture and incubated for 5min on ice. The trypsinization was stopped by washing using DMEM and 10%FBS. The fibroblasts were then collected by pipetting and spun down in acentrifuge at 1000 g for 5 min.

Fibroblasts were resuspended in PBS with 1% Low Melting Point Agarose(LMP) and 100 μl was poured into agarose plug molds. Once solidified,the agarose plugs were extracted from their molds and transferred into a50 ml tube containing the 500 μl of lysis buffer per plug, i.e. 1 mg/mlProteinase K (NEB) in 0.5M EDTA and 1% N-lauroylsarcosine, freshlyprepared. The plugs were incubated for 16h at 50° C. with gentleagitation. The plugs were then washed with 50 mM EDTA and stored in 50mM EDTA at 4° C.

To recover the DNA, the agarose plug was first melted at 68° C. in 1×agarase buffer (NEB) for 10 minutes and then cooled down to 42° C. andincubated for 2h at 42° C. with 1 unit of beta-agarase I (NEB) per 100μl volume. The enzyme was then heat-denatured at 68° C. for 10 minutes.Megabases long double-stranded DNA fragments were ready formanipulation.

Example II

The megabase long DNA was diluted in 100 mM MES ph 5.5 (Sigma), whichfavors attachment of the DNA 5′ termini to a silanated surface. A glassmicroscope cover slip or a piece of silicon was treated with 1%vinylsilane (Sigma) and then rinsed with distilled water before beingattached by a clip to a combing device and then dipped in a reservoircontaining 200 μl of diluted DNA in MES. The reservoir has the properdimension to minimize the volume while allowing the cover slip to becompletely dipped into the liquid in the reservoir. The combing deviceincluded a syringe pump (Tecan model #737472), with the clip attached tothe pump vertical axis, and the vertical motion of the pump was adjustedvia computer software, and typically set at 250 μm/s.

The cover slip was pulled from the reservoir, at a constant speed, whichresulted in combing of the DNA at the meniscus. Once the cover slip wascompletely pulled-out from the reservoir containing DNA, it was rinsedwith 2×SSC (300 mM sodium chloride, 30 mM trisodium citrate, pH 7.0) andassembled into a flow cell by superposing a clean microscope glass slidewith a gasket in between to allow liquid flowing from one extremity tothe other. FIG. 5 is an image of stretched DNA stained with YOYO-1 usingfluorescent microscopy (blue channel) at 320× magnification. As shown inFIG. 5, megabase long DNA was stretched using the method described inExample II. If hybridization and ligation are to be performed, then theflow cell/slide may be washed with a solution containing 100 μg/mlBovine Serum Albumin (BSA, NEB) to block the remaining binding sites.For example, after stretching the DNA, the slide may be washed with SSC,then washed with BSA and then washed with SSC.

Example III

While dsDNA can easily be linearized and deposited on a surface byseveral methods including molecular combing, a single-stranded nucleicacid fragment is typically preferred for interrogation usinghybridization or ligation.

In one embodiment, dsDNA is rendered single stranded with the singlestrand attached to the cover slip by contacting the cover slip assembledinto the flow cell with a denaturing solution containing 0.1N sodiumhydroxide, or 100% formamide or 100% DMSO. The cover slip was contactedseveral times with the denaturing solution. Since the 5′ termini of eachDNA strand is preferentially bound to the vinylsilane-coated cover slip,the complementary DNA strand was displaced by the parallel flow of thedenaturant. The denaturant is removed by washing using 2×SSC.

In another embodiment, one strand of the dsDNA was randomly nicked usingthe property of DnaseI to only partially digest the strand in theabsence of manganese or magnesium ions. Briefly, 1 unit of DnaseI (NEB)was added to 100 μl of a 10 mM Tris-HCL pH 7.5 solution, and incubatedand loaded in the flow cell containing the linearized DNA and incubatedfor 10 minutes at RT. Then it was washed extensively with 10 mMTris-EDTA. Then 1 unit of Mung Bean nuclease (NEB) in 100 μl 1× MungBean Nuclease Buffer (NEB) was loaded into the flow cell and incubatedat room temperature for 1 minute then washed extensively with 10 mMTris-EDTA. This method creates partially digested and overlapping ssDNAfragments with a 5′phosphate and 3′hydroxyl groups, which are compatiblewith DNA ligation.

Example IV

The DNA Origami structures were designed using the software cadnano2(see cadnano.org; Douglas et al., Nuc. Acids Res., vol. 37, no. 15, pp.5001-5006 hereby incorporated by reference in its entirety). By example,a 100 nm long barrel shape can be designed, which also contains freessDNA regions that will be used as binding sites to label the Origamiwith fluorescent oligo probe. FIG. 6 is a three dimensional CADrepresentation of a barrel-shaped DNA origami, with a hanging ssDNAbinding site extending off of the barrel, which was designed usingcadnano2. A list of oligo-staples were designed and submitted to IDT forsynthesis.

The 3D origami structures where folded according to Douglas et al.,Nature, 459(7245): 414-418. doi:10.1038/nature08016 (2009) incorporatedby reference herein in its entirety. 100 nM of the staples were mixedwith 20 nM of M 13mp18, used as a scaffold DNA, in Origami foldingbuffer (5 mM Tris-EDTA with 16 mM Magnesium Chloride), in a 0.2 ml PCRtube. The DNA in the scaffold-staples was first denatured in a PCRmachine at 95° C. for 10 seconds, then cooled down to 85° C. at a rateof 5° C. per 5 minutes, then to 70° C. at 1° C./5 min, then to 25° C. at1° C./10 min. The reaction typically achieved above 95% foldingefficiency. Removing the excess staples was done by filtering through anAmicon Ultra 100 kDa column (Millipore).

Following DNA origami folding, the DNA origami structures can be labeledwith one of many DNA staining dyes, particles, nanoparticles, metals, orvia fluorescently labeled oligonucleotide probes specific to eachOrigami (1 μl of 25 μM oligo-probe mix in 2×SSC). The Origami isfiltered using an Amicon Ultra 100 kDa column (Millipore) using Origamifolding buffer to equilibrate, wash and elute.

Example V

Labeled Origami probes were mixed in an equimolar ratio. 100 μL ofligation mix containing 1 μM of Origami-probe mix and 2000U of T4 DNAligase in 1×DNA Ligase buffer (Enzymatics) was loaded in the flow celland incubated at room temperature for 30 min. The unligatedOrigami-probes were washed using 500 μL of 2×SSC.

Example VI

After hybridization, ligation and washes, the Origami bound to thetemplate can be identified several ways. Fluorescently labeled Origamican be visualized using an epifluorescent microscope (e.g. LeicaDM16000B) equipped with the appropriate objective (e.g. 20× PLAN APO),and filter sets (e.g. to detect Cy3, a filter block equipped with a545/15 nm bandpass excitation filter, a 610/38 nm bandpass emissionfilter, and a 570 nm longpass dichromatic mirror is used). Thefluorescent signal is indicative of the Origami sequence, which is thenuse to infer the template sequence.

FIG. 7 is an image of Origami probes bound to an aminosilane-coatedcover slip in the absence of template, captured by fluorescentmicroscopy at 320× magnification and blue, green and red channelssuperposed.

FIGS. 8A-8C are images of a set of stretched DNA templates, bound to avinylsilane-coated coverslip, assembled to a flow cell, and then probedwith fluorescently labeled Origami. The image was captured byfluorescent microscopy at 320× magnification. Top left: Green channel(CY3), top right: Red channel (CY5), bottom center: superposition ofboth channels.

FIG. 9 are images of stretched DNA template, bound to avinylsilane-coated coverslip, assembled to a flow cell, and then probedwith a set of fluorescently labeled Origami, each specific to adifferent nucleic acid base, using T4 DNA ligase. The image was capturedby fluorescent microscopy at 320× magnification. From left to right:Blue channel (FAM), Green channel (CY3), Red channel (CY5), andsuperposition of all three channels.

At higher resolution, by using super-resolution microscopy (NikonSTORM), a given Origami is labeled with multiple oligo-probes, creatinga color barcode. This color code can be resolved to provide informationabout the sequence.

In another method, the linearized template is laid on a carbon grid touse with an electron microscope (JEOL JEM-1400), which provides theresolution to identify the Origami based on its shape, and then identifythe nucleic acid sequence to which it corresponds. In this case, theOrigami are labeled with 10 nm gold nanoparticles (AuNP) tagged witholigonucleotide complementary to the origami sequence. AuNP are firstcoated with 25 mM Phosphine (BSPP, Sigma) and are incubated overnightwith gentle mixing. A 5M Sodium Chloride solution is then slowly addedto the reaction until it turns to light purple. 100 μM Thiolated-oligo(IDT) were incubated in 20 mM TCEP (Sigma) for 30 minutes and purifiedusing an Amicon Ultra 10 kDa column (Millipore). 100 μM oligo and 1 μMAuNP were then conjugated in Origami folding buffer (5 mM Tris-EDTA with16 mM Magnesium Chloride) overnight, and purified in an Amicon Ultra 100kDa column. 1 μL of 1 μM AuNP-DNA was added per 100 nM Origami andincubated overnight with gentle mixing. TEM images were generatedfollowing JEOL recommendation, typically using high voltage (100 kV).

1. A method for determining the sequence of nucleotides in a singlestranded nucleic acid comprising imaging the single stranded nucleicacid having an oligonucleotide probe hybridized thereto, wherein theoligonucleotide probe includes a spatially distinct nucleic acidstructure corresponding to one or more nucleotides in theoligonucleotide probe, and identifying the spatially distinct nucleicacid structure, the corresponding one or more nucleotides in theoligonucleotide probe and a complementary one or more nucleotides in thesingle stranded nucleic acid.
 2. The method of claim 1 wherein thesingle stranded nucleic acid has a plurality of oligonucleotide probeshybridized thereto and wherein each spatially distinct nucleic acidstructure, the one or more corresponding nucleotides in theoligonucleotide probe and the one or more complementary nucleotides inthe single stranded nucleic acid are identified.
 3. The method of claim1 wherein the oligonucleotide probe is ligated to a sequencing primerhybridized to the single stranded nucleic acid.
 4. The method of claim 1wherein the single stranded nucleic acid is straightened.
 5. The methodof claim 1 wherein the oligonucleotide probe is a nucleic acid sequencehaving between about 1 and about 100 hybridizable nucleotides.
 6. Themethod of claim 1 wherein the spatially distinct nucleic acid structureincludes a detectable label corresponding to the one or more nucleotidesof the hybridized oligonucleotide probe and the detectable label isdetected.
 7. The method of claim 1 wherein the spatially distinctnucleic acid structure is a DNA origami.
 8. A method for determining thesequence of nucleotides in a single stranded nucleic acid comprisinghybridizing an oligonucleotide probe having a spatially distinct nucleicacid corresponding to one or more nucleotides in the oligonucleotideprobe, imaging the single stranded nucleic acid having theoligonucleotide probe hybridized thereto, and identifying the spatiallydistinct nucleic acid, the corresponding one or more nucleotides in theoligonucleotide probe and a complementary one or more nucleotides in thesingle stranded nucleic acid.
 9. The method of claim 8 further includinghybridizing a plurality of oligonucleotide probes along the singlestranded nucleic acid, imaging the single stranded nucleic acid havingthe oligonucleotide probes hybridized thereto, and identifying thespatially distinct nucleic acid structures, the corresponding one ormore nucleotides in the oligonucleotide probe and a complementary one ormore nucleotides in the single stranded nucleic acid.
 10. The method ofclaim 8 wherein the oligonucleotide probe is ligated to a sequencingprimer hybridized to the single stranded nucleic acid.
 11. The method ofclaim 8 wherein the single stranded nucleic acid is straightened. 12.The method of claim 8 wherein the oligonucleotide probe is a nucleicacid sequence having between about 1 and about 100 hybridizablenucleotides.
 13. The method of claim 8 wherein the spatially distinctnucleic acid structure includes a detectable label corresponding to theone or more nucleotides of the hybridized oligonucleotide probe and thedetectable label is detected.
 14. The method of claim 8 wherein thespatially distinct nucleic acid structure is a DNA origami.
 15. A methodfor determining the sequence of nucleotides in a single stranded nucleicacid comprising hybridizing an oligonucleotide probe having a spatiallydistinct nucleic acid corresponding to one or more nucleotides in theoligonucleotide probe, ligating the oligonucleotide probe to asequencing primer hybridized to the single stranded nucleic acid,imaging the single stranded nucleic acid having the oligonucleotideprobe hybridized and ligated thereto, and identifying the spatiallydistinct nucleic acid, the corresponding one or more nucleotides in theoligonucleotide probe and a complementary one or more nucleotides in thesingle stranded nucleic acid.
 16. The method of claim 15 furtherincluding hybridizing a plurality of oligonucleotide probes along thesingle stranded nucleic acid, imaging the single stranded nucleic acidhaving the oligonucleotide probes hybridized thereto, and identifyingthe spatially distinct nucleic acid structures, the corresponding one ormore nucleotides in the oligonucleotide probe and a complementary one ormore nucleotides in the single stranded nucleic acid.
 17. The method ofclaim 15 wherein the oligonucleotide probe is ligated to a sequencingprimer hybridized to the single stranded nucleic acid.
 18. The method ofclaim 15 wherein the single stranded nucleic acid is straightened. 19.The method of claim 15 wherein the oligonucleotide probe is a nucleicacid sequence having between about 1 and about 100 hybridizablenucleotides.
 20. The method of claim 15 wherein the spatially distinctnucleic acid structure includes a detectable label corresponding to theone or more nucleotides of the hybridized oligonucleotide probe and thedetectable label is detected.
 21. The method of claim 15 wherein thespatially distinct nucleic acid structure is a DNA origami.
 22. A methodfor determining the sequence of nucleotides in a single stranded nucleicacid comprising hybridizing an oligonucleotide probe having a spatiallydistinct nucleic acid corresponding to one or more nucleotides in theoligonucleotide probe, ligating the oligonucleotide probe to asequencing primer hybridized to the single stranded nucleic acid,straightening the single stranded nucleic acid having theoligonucleotide probe hybridized and ligated thereto, imaging the singlestranded nucleic acid having the oligonucleotide probe hybridized andligated thereto, and identifying the spatially distinct nucleic acid,the corresponding one or more nucleotides in the oligonucleotide probeand a complementary one or more nucleotides in the single strandednucleic acid.
 23. The method of claim 22 further including hybridizing aplurality of oligonucleotide probes along the single stranded nucleicacid, imaging the single stranded nucleic acid having theoligonucleotide probes hybridized thereto, and identifying the spatiallydistinct nucleic acid structures, the corresponding one or morenucleotides in the oligonucleotide probe and a complementary one or morenucleotides in the single stranded nucleic acid.
 24. The method of claim22 wherein the oligonucleotide probe is ligated to a sequencing primerhybridized to the single stranded nucleic acid.
 25. The method of claim22 wherein the single stranded nucleic acid is straightened.
 26. Themethod of claim 22 wherein the oligonucleotide probe is a nucleic acidsequence having between about 1 and about 100 hybridizable nucleotides.27. The method of claim 22 wherein the spatially distinct nucleic acidstructure includes a detectable label corresponding to the one or morenucleotides of the hybridized oligonucleotide probe and the detectablelabel is detected.
 28. The method of claim 22 wherein the spatiallydistinct nucleic acid structure is a DNA origami.
 29. A method fordetermining the sequence of nucleotides in a single stranded nucleicacid comprising hybridizing an oligonucleotide probe having a spatiallydistinct nucleic acid corresponding to one or more nucleotides in theoligonucleotide probe, straightening the single stranded nucleic acidhaving the oligonucleotide probe hybridized thereto, imaging the singlestranded nucleic acid having the oligonucleotide probe hybridizedthereto, and identifying the spatially distinct nucleic acid, thecorresponding one or more nucleotides in the oligonucleotide probe and acomplementary one or more nucleotides in the single stranded nucleicacid.
 30. The method of claim 29 further including hybridizing aplurality of oligonucleotide probes along the single stranded nucleicacid, imaging the single stranded nucleic acid having theoligonucleotide probes hybridized thereto, and identifying the spatiallydistinct nucleic acid structures, the corresponding one or morenucleotides in the oligonucleotide probe and a complementary one or morenucleotides in the single stranded nucleic acid.
 31. The method of claim29 wherein the oligonucleotide probe is ligated to a sequencing primerhybridized to the single stranded nucleic acid.
 32. The method of claim29 wherein the oligonucleotide probe is a nucleic acid sequence havingbetween about 1 and about 100 hybridizable nucleotides.
 33. The methodof claim 29 wherein the spatially distinct nucleic acid structureincludes a detectable label corresponding to the one or more nucleotidesof the hybridized oligonucleotide probe and the detectable label isdetected.
 34. The method of claim 29 wherein the spatially distinctnucleic acid structure is a DNA origami.
 35. A method for determiningthe sequence of nucleotides in a single stranded nucleic acid comprisingstraightening the single stranded nucleic acid, hybridizing to thesingle stranded nucleic acid an oligonucleotide probe having a spatiallydistinct nucleic acid corresponding to one or more nucleotides in theoligonucleotide probe, imaging the single stranded nucleic acid havingthe oligonucleotide probe hybridized thereto, and identifying thespatially distinct nucleic acid, the corresponding one or morenucleotides in the oligonucleotide probe and a complementary one or morenucleotides in the single stranded nucleic acid.
 36. The method of claim35 further including hybridizing a plurality of oligonucleotide probesalong the single stranded nucleic acid, imaging the single strandednucleic acid having the oligonucleotide probes hybridized thereto, andidentifying the spatially distinct nucleic acid structures, thecorresponding one or more nucleotides in the oligonucleotide probe and acomplementary one or more nucleotides in the single stranded nucleicacid.
 37. The method of claim 35 wherein the oligonucleotide probe isligated to a sequencing primer hybridized to the single stranded nucleicacid.
 38. The method of claim 35 wherein the oligonucleotide probe is anucleic acid sequence having between about 1 and about 100 hybridizablenucleotides.
 39. The method of claim 35 wherein the spatially distinctnucleic acid structure includes a detectable label corresponding to theone or more nucleotides of the hybridized oligonucleotide probe and thedetectable label is detected.
 40. The method of claim 35 wherein thespatially distinct nucleic acid structure is a DNA origami.
 41. A methodfor determining the sequence of nucleotides in a single stranded nucleicacid comprising straightening the single stranded nucleic acid,hybridizing to the single stranded nucleic acid an oligonucleotide probehaving a spatially distinct nucleic acid corresponding to one or morenucleotides in the oligonucleotide probe, ligating the oligonucleotideprobe to a sequencing primer hybridized to the single stranded nucleicacid, imaging the single stranded nucleic acid having theoligonucleotide probe hybridized and ligated thereto, and identifyingthe spatially distinct nucleic acid, the corresponding one or morenucleotides in the oligonucleotide probe and a complementary one or morenucleotides in the single stranded nucleic acid.
 42. The method of claim41 further including hybridizing and ligating a plurality ofoligonucleotide probes along the single stranded nucleic acid, imagingthe single stranded nucleic acid having the oligonucleotide probeshybridized and ligated thereto, and identifying the spatially distinctnucleic acid structures, the corresponding one or more nucleotides inthe oligonucleotide probe and a complementary one or more nucleotides inthe single stranded nucleic acid.
 43. The method of claim 41 wherein theoligonucleotide probe is a nucleic acid sequence having between about 1and about 100 hybridizable nucleotides.
 44. The method of claim 41wherein the spatially distinct nucleic acid structure includes adetectable label corresponding to the one or more nucleotides of thehybridized oligonucleotide probe and the detectable label is detected.45. The method of claim 41 wherein the spatially distinct nucleic acidstructure is a DNA origami.
 46. An oligonucleotide probe comprising atemplate-hybridizing nucleic acid structure and a spatially distinctnucleic acid structure corresponding to one or more nucleotides in thetemplate-hybridizing nucleic acid structure.
 47. The oligonucleotideprobe of claim 46 wherein the spatially distinct nucleic acid structureincludes a detectable moiety.
 48. The oligonucleotide probe of claim 46wherein the template-hybridizing nucleic acid structure includes betweenabout 1 nucleotide to about 100 nucleotides.
 49. The oligonucleotideprobe of claim 46 wherein the template-hybridizing nucleic acidstructure includes 6 nucleotides.
 50. The oligonucleotide probe of claim46 wherein the spatially distinct nucleic acid structure is a DNAorigami.